FI92326C - LHRH nonapeptide and decapeptide analogs useful as LHRH antagonists - Google Patents
LHRH nonapeptide and decapeptide analogs useful as LHRH antagonists Download PDFInfo
- Publication number
- FI92326C FI92326C FI880509A FI880509A FI92326C FI 92326 C FI92326 C FI 92326C FI 880509 A FI880509 A FI 880509A FI 880509 A FI880509 A FI 880509A FI 92326 C FI92326 C FI 92326C
- Authority
- FI
- Finland
- Prior art keywords
- phe
- pal
- nal
- pro
- leu
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/23—Luteinising hormone-releasing hormone [LHRH]; Related peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S930/00—Peptide or protein sequence
- Y10S930/01—Peptide or protein sequence
- Y10S930/13—Luteinizing hormone-releasing hormone; related peptides
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Endocrinology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
- Fodder In General (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
Description
5 >. 3 2 6 LHRH:n nonapeptidi- ja dekapeptidianalogeja, jotka ovat kåyttdkelpoisia LHRH:n antagonisteina5>. 3 2 6 LHRH nonapeptide and decapeptide analogues useful as LHRH antagonists
Luteinisoivaa hormonia (LH) ja follikkelia stimu-5 loivaa hormonia (FSH) vapautuu aivolisSkkeen etulohkosta hypothalamuksen alueella tuotettavan luteinisoivaa hormonia vapauttavan hormonin (LHRH, LH/FSH-RH, GnRH) saate-lyn alaisena. LH ja FSH vaikuttavat gonadeihin kiihdyt-tSen steroidihormonien synteesia ja gameettien kypsymis-10 ta. LHRH:n sykayksittåinen vapautuminen ja siten LH:n ja FSH:n vapautuminen saatelevat lisaantymissykliå kotieiai-milia ja ihmisilia.Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are released from the anterior lobe of the brain under the control of luteinizing hormone releasing hormone (LHRH, LH / FSH-RH, GnRH) produced in the hypothalamic region. LH and FSH affect gonads by accelerating the synthesis of steroid hormones and the maturation of gametes. The single release of LHRH and thus the release of LH and FSH accompany the reproductive cycle in domestic and human cilia.
LHRH vaikuttaa myiis istukkaan ja sita kautta epa-suorasti gonadeihin aikaansaamalla koriogonadotropiinin 15 (OG) synteesin ja vapautumisen.LHRH also affects the placenta and through it epa-directly to the gonads by inducing the synthesis and release of choriogonadotropin 15 (OG).
LHRH:n antagonistit ovat kayttdkelpoisia hedelmai-lisyyden saatelyssa. Sellaiset antagonistit estavat ovu-laatiota naarailla ja spermatogeneesia uroksilla. Naihin vaikutuksiin liittyy laheisesti gonadiperåisten steroi-• 20 disten sukupuolihormonien tason aleneminen verenkierros-sa esiintyvasta normaalitasosta, mika aiheuttaa aksesso-risten elinten massan pienentymista uroksilla ja naarailla. Kotielaimilia tama vaikutus ehkaisee sukupuolista kiertoa ja kayttaytymista (edistaen painon lisåantymista ..25 laiduntamisvaiheessa), aiheuttaa keskenmenoja tiineilia I « eiaimilia ja saa yleensa aikaan kemiallisen sterilisaa-tion.LHRH antagonists are useful in fertilization. Such antagonists inhibit ovulation in females and spermatogenesis in males. These effects are closely related to a decrease in the level of gonad-derived steroid sex hormones from normal circulatory levels, which causes a decrease in accessory organ mass in males and females. In domestic animals, this effect prevents sexual rotation and behavior (promoting weight gain ..25 during the grazing phase), causes miscarriages of pregnant women and usually results in chemical sterilization.
Luonnollinen vapauttava hormoni LHRH on dekapep-tidi, joka koostuu luonnossa esiintyvista aminohapoista 30 (joilla glysiinia, joka on asymmetriakeskukseton amino-·;· happo, lukuunottamatta on L-konfiguraatio). Sen aminohap- pojarjestys on seuraava: (pyro) Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH^ 35 123456789 10 2 ?V7"6 Tåmån luonnollisen aineen kanssa analogiset yhdisteet esitetåan usein lyhennetysså muodossa osoittamalla jon-kin tietyn aminohapon, jonka asema osoitetaan ylåindek-sillå, korvaajan luonne, mitå seuraa "LHRH". Monia LHRH:n 5 analogeja on tutkittu, ja hyvin suurella enemmistollå niista on riittåmåton biologinen aktiivisuus, jotta ne olisivat kliinisesti kayttokelpoisia. Eråillå tietyilla muunnoksilla on kuitenkin agonistista biologista aktii-visuutta tehostava vaikutus. Huomattava agonistisen ak-10 tiivisuuden lisaåntyminen saavutetaan vaihtamalla Gly:n tilalle 6-asemassa sijaitsevaksi happotahteeksi D-ami-nohappotahde.The natural releasing hormone LHRH is a decapeptide composed of naturally occurring amino acids 30 (with glycine, which is an asymmetric-centered amino acid; except for the L-configuration). Its amino acid sequence is as follows: (pyro) Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH 4 35 123456789 10 2? V7 "6 Compounds analogous to this natural substance are often shown in abbreviated form by demonstrating the nature of the substitute for a particular amino acid whose position is indicated by a superscript, followed by “LHRH.” Many analogs of LHRH have been studied, and a very large majority of them have insufficient biological activity to be clinically useful. however, the modifications have the effect of enhancing agonistic biological activity A significant increase in agonist activity is achieved by replacing Gly with an D-amino acid residue at the 6-position acid residue.
Agonistien lisaksi on valmistettu analogeja, jot-ka ovat LHRH:n kompetitiivisia antagonisteja ja jotka 15 kaikki edellyttåvat 2-asemassa sijaitsevan histidyyli-ryhmån poistamista tai korvaamista Zw· Vale et al., Science 176 (1972) 933/. Yleisesti ottaen vaikuttaa sil-ta, etta D-aminohappotåhde tuottaa kyseiseen asemaan ketjussa sijoitettuna tulokseksi parhaan aktiivisuuden 20 ZR.W.A. Rees et al., J.Med.Chem. 17 (1974) 10167-In addition to agonists, analogs have been prepared which are competitive antagonists of LHRH and all of which require the removal or replacement of the histidyl group in the 2-position Zw · Vale et al., Science 176 (1972) 933 /. In general, a D-amino acid residue appears to produce the best activity at 20 ZR.W.A when placed at that position in the chain. Rees et al., J. Med. Chem. 17 (1974) 10167-
Sen lisaksi lisamuunnos 6-asemassa (joka ilman 2-aseman muuntamista johtaa edella mainittuun agonisti-seen aktiivisuuteen) lisaå 2-muunnettujen analogien an-tagonistista aktiivisuutta iC. W. Beattie et al., J. Med.In addition, further modification at the 6-position (which, without 2-position conversion, results in the aforementioned agonist activity) increases the antagonist activity iC of the 2-modified analogs. W. Beattie et al., J. Med.
·. 25 Chem. 18 (1975) 1247; J. Rivier et al., Peptides 1976, Editions de l'Universite de Bruxeller, Belgia, 1976, s 4277.·. 25 Chem. 18 (1975) 1247; J. Rivier et al., Peptides 1976, Editions de l'Universite de Bruxeller, Belgium, 1976, p. 4277.
Nåiden kahden påamuunnoksen, jotka johtavat te-hokkaampiin LHRH:n antagonisteihin, pohjalta antagonis-30 tista aktiivisuutta on voitu lisata edelleen muuntamalla jo 2,6-muunnetun peptidin 1-, 3-, 5- ja/tai 10-asemaa 7¾. H. Coy et al., Peptides 1976, Editions de l'Universite de Bruxelles, Belgia, 1976, s 462; J. E. Rivier et al., Life Sci. 23 (1978) 869; A. S. Dutta et al., 35 Biochem. Biophys. Res. Commun. 85 (1979) 7097· 0n mySs 1On the basis of these two main variants, which lead to more potent LHRH antagonists, the antagonistic activity has been further increased by converting the 1-, 3-, 5- and / or 10-position 7 jo of the already 2,6-modified peptide. H. Coy et al., Peptides 1976, Editions de l'Universite de Bruxelles, Belgium, 1976, s 462; J. E. Rivier et al., Life Sci. 23 (1978) 869; A. S. Dutta et al., 35 Biochem. Biophys. Res. Commun. 85 (1979) 7097 · 0n MySs 1
IIII
· 3 i? 2 3 2 6 osoitettu, etta 1-asemassa sijaitsevan aminohappotåhteen N-asyloinnista on etua /K. Channabasavaia et al., Bio-chem. Biophys. Res. Commun. 81 (1978) 382; D. H. Coy et al., Peptides - Structure and Biological Function, Pier-5 ce Chemical Co., 1979, s 775/. Lisåksi on julkaistu ar-tikkeli /D.H. Coy, Endocrinology 110 (1982) 1445/1 joka kasittelee erittain tehokasta antagonistia, joka sisal- r i p taa D-Arg -substituution, (N-Ac-D-pCl-Phe , D-pCl-Phe , D-Trp3, D-Arg6, D-Ala10)LHRH.· 3 i? 2 3 2 6 shown that N-acylation of an amino acid residue at position 1 has the advantage / K. Channabasavaia et al., Bio-chem. Biophys. Res. Commun. 81 (1978) 382; D. H. Coy et al., Peptides - Structure and Biological Function, Pier-5 ce Chemical Co., 1979, p. 775 /. In addition, the article /D.H. Coy, Endocrinology 110 (1982) 1445/1 which deals with a highly potent antagonist containing the D-Arg substitution, (N-Ac-D-pCl-Phe, D-pCl-Phe, D-Trp3, D- Arg6, D-Ala10) LHRH.
β 10 Ikava kyllå, vaikka D-Arg -substituution sisaltava rynmå LHRH:n analogeja on havaittu tehokkaiksi ovulaa- tiota estaviksi aineiksi, ne olivat myos voimakkaita syottSsoluja degranuloivia aineita /Schmidt et al.,β 10 Yes, although DHR-containing analogs of LHRH containing the D-Arg substitution have been found to be effective anti-ovulatory agents, they were also potent cells. Cell degranulants / Schmidt et al.,
Contraception 29 (1984) 283/ ja aiheuttivat odeemaa in 1 2 15 vivo. Niinpa esimerkiksi (N-Ac-D-Nal(2) , D-pCl-Phe , -¾ β D-Trp , D-Arg )LHRH:n ED,-g on 0,2 yug/ml, mita tulee his-tamiinin vapauttamiseen rotan syottosoluista in vitro.Contraception 29 (1984) 283 / and caused eczema in 1 2 15 vivo. Thus, for example (N-Ac-D-Nal (2), D-pCl-Phe, -¾ β D-Trp, D-Arg), the ED, -g of LHRH is 0.2 μg / ml, which becomes for the release of amine from rat mast cells in vitro.
Tama sivureaktio on kliinisesti tårkeS seuraavan anafy-laktistyyppisen reaktion mahdollisen hengenvaarallisen 20 luonteen vuoksi.This side reaction is clinically important due to the potentially life-threatening nature of the following anaphylactic-type reaction.
On alalla tunnettua, etta positiivisen varauksen/-positiivisia varauksia, erityisesti useita positiivisia varauksia, joihin yhdistyy hydrofobisuus, sisaltavat mo-lekyylit ovat voimakkaita syottosolujen degranuloijia '1 25 /Foreman ja Jordan, Agents and Action 13 (1983) 1057· Ensimmainen yritys kiertaa tamå ongelma kaksi Arg-tah-detta (asemissa 6 ja 8) sisSltavien analogien yhteydes-sa oli lisata tShteiden etSisyyttå (esim. (N-Ac-D-Nal(2)1, D-pCl-Phe2, D-Trp3, Arg5, D-Tyr6, D-Ala10)LHRH; ED^Q = 30 2yug/ml histamiinin vapautuksessa; R.W. Roeske et al., • 5 5 "Substitution of Arg for Tyr in GNRH antagonists".It is known in the art that molecules containing positive charge / positive charges, especially multiple positive charges combined with hydrophobicity, are potent mast cell degranulators' 1 25 / Foreman and Jordan, Agents and Action 13 (1983) 1057 · The first attempt to circumvent this the problem with analogs containing two Arg-strokes (at positions 6 and 8) was to increase the searchability of the compounds (e.g., N-Ac-D-Nal (2) 1, D-pCl-Phe2, D-Trp3, Arg5, D-Tyr6, D-Ala10) LHRH; ED ^ Q = 30 2ug / ml in histamine release; RW Roeske et al., • 5 "Substitution of Arg for Tyr in GNRH antagonists".
Peptides - Structure and Function, toim. C.M. Deber, V. J. Hruby ja K. D. Koppie, Pierce Chemical Co.,Peptides - Structure and Function, ed. C.M. Deber, V. J. Hruby and K. D. Koppie, Pierce Chemical Co.,
Rockford, Illinois, 1985, s 561). Vaikka tama heikensi 35 analogin kykyå degranuloida syottosoluja ja vapauttaa * 4 9/326 histamiinia, analogilla oli yha monta kertaa suurempi ky-ky aiheuttaa anafylaktistyyppinen reaktio kuin LHRH:11a, jonka EDgg on 328 pg/ml, mita tulee histamiinin vapautta-miseen.Rockford, Illinois, 1985, p. 561). Although this impaired the ability of 35 analogs to degranulate mast cells and release * 4 9/326 histamine, the analog had an increasing number of times the ability to elicit an anaphylactic-type reaction than LHRH with an EDgg of 328 pg / ml in terms of histamine release.
5 Mytts Lys(iPr) on sisailytetty eri asemiin LHRH:n antagonisteihin. Sijoitettaessa se 6- ja 8-asemaan, kun 2-asemassa on samanaikaisesti D-pCl-Phe-tåhde, suuri ovu-laation vastainen teho ja heikentynyt histamiinin vapau-tuminen såilyvat (esim. (N-Ac-D-Nal(2)\ pCl-Phe2, q o in 10 D-Trp , D-Lys(iPr) , D-Ala )LHRH; EDj-q = 6,6 pg/ml histamiinin vapautuksessa). ErSåseen analogiin sijoitettiin 8-asemaan hArg(Et-), jolloin analogin kyky vapauttaa his- z 1 tamiinia oli samaa tasoa (so. (N-Ac-D-Nal(2) , D-oMe-pCl-Phe2, D-Pal(3)3, D-Arg6, hArg(Et^)8, 10 z 15 D-Ala )LHRH; ED^q =4,9 pg/ml histamiinin vapautuksessa). Voidaan kuitenkin havaita, etta nåmå analogit ovat silti hyvin tehokkaita histamiinia vapauttavia aineita LHRH:hon verrattuna. (R.W. Roeske et al., "LHRH Antagonists with Low Histamine Releasing Activity". LHRH and 20 its Analogs: Contraceptive and Therapeutic Applications, Part 2, toim. B. H. Vickery ja J. J. Nestor Jr., MTP Press, Boston, 1987, s 17-24).5 Mytts Lys (iPr) is included at various positions in LHRH antagonists. When placed in the 6- and 8-positions with a simultaneous D-pCl-Phe residue in the 2-position, high anti-ovulation potency and decreased histamine release are maintained (e.g., N-Ac-D-Nal (2)). pCl-Phe2, qo in 10 D-Trp, D-Lys (iPr), D-Ala) LHRH; EDj-q = 6.6 pg / ml in histamine release). The ErSåse analog was placed in the 8-position hArg (Et-), whereby the ability of the analog to release his z 1 amine was at the same level (i.e., N-Ac-D-Nal (2), D-oMe-pCl-Phe2, D-Pal). (3) 3, D-Arg6, hArg (Et 2 O) 8, 10 z 15 D-Ala) LHRH (ED 2 q = 4.9 pg / ml in histamine release). However, it can be seen that these analogs are still very effective histamine releasing agents compared to LHRH. (RW Roeske et al., “LHRH Antagonists with Low Histamine Releasing Activity.” LHRH and its Analogs: Contraceptive and Therapeutic Applications, Part 2, edited by BH Vickery and JJ Nestor Jr., MTP Press, Boston, 1987, p. 17). -24).
8- ja 9-asema on myOs tunnistettu tarkeimmiksi substituoitaviksi kohdiksi, jotta estetaan histamiinin - 25 vapautus, koska Arg-Pro-jakso on neuropeptideissa, jotka aiheuttavat syOttGsolujen degranuloitumista, usein ha-vaittu ominaispiirre. Vaikka siis 8-asema on tieteelli-sin perustein ratkaiseva asema substituoitavaksi, nykyi-sin tunnetulla joukolla analogeja on huomattava mahdolli-30 suus myrkyllisyyteen ja muihin sivuvaikutuksiin.The 8- and 9-positions have also been identified as the most important substitutable sites to inhibit histamine-25 release, as the Arg-Pro sequence is a frequently observed feature in neuropeptides that cause degranulation of feed cells. Thus, although the 8-position is, on a scientific basis, a crucial position for substitution, a number of analogs currently known have a significant potential for toxicity and other side effects.
Tama keksintd koskee uusia, hyvin tehokkaita LHRHrn nonapeptidi- ja dekapeptidianalogeja, joiden kyky vapauttaa histamiinia on hyvin pieni ja joiden ratkaise-van tarkea ominaispiirre on steerisesti estetty guanidi-35 nosubstituoitu arginyyliryhma 8-asemassa, vaittaen argi-nyyliryhmaa 6-asemassa. KeksintO koskee myOs koostumuk-j sia, jotka sisaltavat naita yhdisteita.This invention relates to novel, highly potent nonapeptide and decapeptide analogs of LHRH that have very low histamine release capacity and that have the sterically hindered guanide-35 nosubstituted arginyl group at the 8-position, with the exception of the Argyl group at the 6-position. The invention relates to myOs compositions containing these compounds.
5 9'/ 32659 '/ 326
Keksinndn kohteena on yhdiste, jolla on kaava (I), A-B-C-Ser-D-E-F-G-Pro-J (I) 123 4 5678 9 10 5 tai sen fysiologisesti hyvåksyttåvå suola.The invention relates to a compound of the formula (I), A-B-C-Ser-D-E-F-G-Pro-J (I) 123 4 5678 9 10 5 or a physiologically acceptable salt thereof.
LHRH:n 2-asemassa sijaitsevan L-histidyyliryhman korvaaminen muuttaa peptidin LHRH:n antagonistiksi. LHRH:ssa 6-asemassa sijaitsevan glysyyliryhmån korvaami-10 nen jollakin Erna mainituista ryhmista lisaa antagonis-tista vaikutusta voimakkaasti. Tassa esitetyt 1-, 2-, 3-, 5-, 7- ja 10-asemassa tehtåvåt korvaukset auttavat edelleen parantamaan antagonistista aktiivisuutta. Subs-tituutio G 8-asemassa alentaa voimakkaasti analogien te-15 hoa histamiinin vapautuksessa, kun 6-aseman Arg on kor-vattu jollakin muulla ryhmaiia, ja on ratkaisevan tarkea ajatellen analogien turvallisuutta.Replacement of the L-histidyl group at the 2-position of LHRH converts the peptide to an LHRH antagonist. Replacement of the glycyl group at the 6-position in LHRH with one of the groups mentioned in Erna greatly increases the antagonistic effect. The substitutions at the 1-, 2-, 3-, 5-, 7-, and 10-positions shown herein further help to enhance antagonist activity. Sub-substitution at the G 8 position greatly reduces the effect of the analogs te-15 on histamine release when the Arg at the 6-position is replaced by another group, and is crucial for the safety of the analogs.
Kuten edelia on esitetty, erilaisista yleisista aminohapoista kSytetSSn tamSn keksinnOn kuvauksen helpot-20 tamiseksi tavanomaisia lyhenteita peptidialalla yleisesti hyvSksytylia tavalla, jota on suositellut IUPAC-IUB Commission on Biochemical Nomenclature (Biochemistry 11 (1972) 1726). Kaikki tassa mainitut peptidijaksot on kir-joitettu noudattaen yleisesti hyvaksyttya tapaa, jossa N-. 25 terminaalinen aminohappo on vasemmalla ja C-terminaalinen aminohappo oikealla.As indicated above, various abbreviations for common amino acids can be used to facilitate the description of the present invention in a manner generally accepted in the peptide art as recommended by the IUPAC-IUB Commission on Biochemical Nomenclature (Biochemistry 11 (1972) 1726). All peptide sequences mentioned herein are written according to the generally accepted manner in which N-. The 25 terminal amino acid is on the left and the C-terminal amino acid is on the right.
Esitetyt lyhenteen tarkoittavat L-aminohappoja, poikkeuksena asymmetriakeskukseton (akiraalinen) amino-happoglysiini ja lisapoikkeuksina ne luonnossa esiinty-30 mattOmat tai esiintyvat aminohapot, jotka ovat akiraali-sia tai joita muussa tapauksessa on merkitty D:lia tai DL:lia.Abbreviations shown refer to L-amino acids, with the exception of asymmetric-centered (achiral) amino acid glycine and, with further exceptions, those naturally occurring or non-naturally occurring amino acids that are achiral or otherwise labeled D or DL.
Tulisi huomata, etta J:n ollessa -NH-C(0)-NH2 C-terminaalinen ryhma on atsaglysinamidiryhma.It should be noted that when J is -NH-C (O) -NH 2, the C-terminal group is an azaglycinamide group.
35 Lyhenne D-Glu(AA) tarkoittaa anisolin ja D-Glu- tahteen valista additiotuotetta, jolloin muodostuu p-met- 6 92326 oksifenyyliketoni (glutamiinihapposivuketjun C-terminaa-liseen paahån), so Glu(pMeO-Ph).The abbreviation D-Glu (AA) means an adduct between anisole and a D-Glutenate to form p-meth-6,9326 oxyphenyl ketone (at the C-terminal end of the glutamic acid side chain), i.e. Glu (pMeO-Ph).
ErfiSt muutkin lyhenteet ovat kåyttOkelpoisia kek-sinnOn kuvauksessa. Tåsså keksinnOsså luonnollisen LHRH-5 peptidin aminohappoja korvataan aminohapoilla, joita ei esiinny luonnossa. Erityisen yleisesti kaytettyja niista ovat seuraavat:Other abbreviations for ErfiSt are also useful in the description of the invention. In this invention, the amino acids of the native LHRH-5 peptide are replaced with amino acids that do not occur in nature. Particularly commonly used are:
Aminohappotahde Lyhenne 3-(2-naftyyli)alanyyli Nal(2) 10 3-(p-fluorifenyyli)alanyyli pF-Phe 3-(p-kloorifenyyli)alanyyli pCl-Phe 3-(3-pyridyyli)alanyyli Pal(3) N,N'-guanidino(dimetyyli)- homoarginyyli Dmh tai hArg(Me)2 15 N,Ν'-guanidino(dietyyli)- homoarginyyli Deh tai hArgCEt^ N,N'-guanidino(diprop- yyli)homoarginyyli Dph tai hArg(Pr>2 N,N'-guanidino(di-iso- 20 propyyli)homoarginyyli Dih tai hArgiiPr^ N,N'-guanidino(diheksyy- li)homoarginyyli Dhh tai hArg(heksyy- 11 >2 N,N'-guanidino(etano)- . . 25 homoarginyyli Eha tai hArgfCI^^ N,N'-guanidino(propeno)- homoarginyyli Pha tai hArgiCl·^)^ N,N'-guanidinobis(2,2,2-tri- fluorietyyli)homoarginyyli Bth tax hArg(CI^CF^)^ 30 NG-etyyli-NG'-(trifluori- etyyli)homoarginyyli hArgiCH^CFg, Et) NG,NG,-2-trifluorimetyyli- 2,2-difluorietyylihomoargi- nyyli hArg(CH2CF2CF3) 35 n-guanidino(etyyli)homo- arginyyli Meh tai hArg(Et) N-guanidino(propyyli)- homoarginyyli Prh tai hArg(Pr) 92326 7Amino acid residue Abbreviation 3- (2-naphthyl) alanyl Nal (2) 10 3- (p-fluorophenyl) alanyl pF-Phe 3- (p-chlorophenyl) alanyl pCl-Phe 3- (3-pyridyl) alanyl Pal (3) N , N'-guanidino (dimethyl) homoarginyl Dmh or hArg (Me) 2 N, Ν'-guanidino (diethyl) homoarginyl Deh or hArgCEt ^ N, N'-guanidino (dipropyl) homoarginyl Dph or hArg (Pr > 2 N, N'-guanidino (diisopropyl) homoarginyl Dih or hArgiiPr ^ N, N'-guanidino (dihexyl) homoarginyl Dhh or hArg (hexyl-> N, N'-guanidino) Homoarginyl Eha or hArgfCl2 ^ N, N'-guanidino (propeno) homoarginyl Pha or hArgiCl · ^) ^ N, N'-guanidinobis (2,2,2-trifluoroethyl) homoarginyl Bth tax hArg (Cl 2 CF 4) ^ 30 NG-ethyl-NG '- (trifluoroethyl) homoarginyl hArgiCH 2 CFg, Et) NG, NG, -2-trifluoromethyl-2,2-difluoroethylhomoarginyl hArg (CH 2 CF 2 CF 3) 35 n- guanidino (ethyl) homoarginyl Meh or hArg (Et) N-guanidino (propyl) homoarginyl Prh or hArg (Pr) 92326 7
AminohappotShde Lyhenne N-guanidino(isopropyyli)- homoarginyyli Tph tai hArg(iPr) N-guanidino(butyyli)homo- 5 arginyyli Mbh tai hArg(Bu) N,N'-guanidino(disyklo- heksyyli)homoarginyyli Dch tai hArg(syklo- heksyyli >2 N-guanidino(heptyyli)homo- 10 arginyyli Hha tai hArg(heptyy li ) N-guanidino(etyyli)arginyyli Mea tai Arg(Et) N,N'-guanidino(di-iso- propyyli)arginyyli Dia tai ArgiiPr^ 15 N,N'-guanidino(disyklo- heksyyli)arginyyli Dca tai Arg(syklohek- syyii)2 3-(3-piperidyyli)alanyyli 3-Pia 3- (4-piperidyyli)alanyyli 4-Pia 20 3-[(NE-metyyli)piperid- 4- yyli]-alanyyli Mpa 3- [(Ne-pentyyli)piperid- 4- yyli]alanyyli Ppa 3-[(Ne-bentsyyli)piperid- — 25 4-yyli]alanyyli BpaAmino AcidsSubject Abbreviation N-guanidino (isopropyl) -homoarginyl Tph or hArg (iPr) N-guanidino (butyl) homo-arginyl Mbh or hArg (Bu) N, N'-guanidino (dicyclohexyl) homoarginyl Dch or hArg (cyclo- hexyl> 2 N-guanidino (heptyl) homo- arginyl Hha or hArg (heptyl) N-guanidino (ethyl) arginyl Mea or Arg (Et) N, N'-guanidino (diisopropyl) arginyl Dia or ArgiiPr N, N'-guanidino (dicyclohexyl) arginyl Dca or Arg (cyclohexyl) -2 3- (3-piperidyl) alanyl 3-Pia 3- (4-piperidyl) alanyl 4-Pia 20 3 - [( NE-methyl) piperid-4-yl] -alanyl Mpa 3 - [(Non-pentyl) piperidin-4-yl] alanyl Ppa 3 - [(N-benzyl) piperid-4-yl] alanyl Bpa
Ne-nikotinyyli-D-lysyyli Lys(Nic) Νε-(3-pyridyyli)asetyyli- D-lysyyli Lys(pyridyyliasetyy- li) 30 3-(2,4,6-trimetyylifenyy- li)alanyyli Tmp 2,2-difenyyliglysyyli Dpg TSssa kaytettynå ilmaisu fysiologisesti hyvaksyt-35 tavat suolat" tarkoittaa suoloja, joilla sailyy kantayh- disteen toivottu biologinen aktiivisuus ja joilla ei ole . mitaan ei-toivottuja toksikologisia vaikutuksia. Esimerk- keja sellaisista suoloista ovat 9 2326 δ (a) epåorgaanisten happojen, esimerkiksi suolahapon, bro-mivetyhapon, rikkihapon, fosforihapon, typpihapon ja vas-taavien, kanssa muodostetut happoadditiosuolat sekå or-gaanisten happojen, kuten esimerkiksi etikkahapon, oksaa- 5 lihapon, viinihapon, meripihkahapon, maleiinihapon, fu- maarihapon, glukonihapon, sitruunahapon, omenahapon, as-korbiinihapon, bentsoehapon, parkkihapon, pamonihapon, algiinihapon, polyglutamiinihapon, naftaleenisulfonihap-pojen, naftaleenidisulfonihappojen ja polygalakturoniha-10 pon, kanssa muodostetut suolat; (b) moniarvoisten metallikationien, kuten sinkin, kalsiu-min, vismutin, bariumin, magnesiumin, alumiinin, kuparin, koboltin, nikkelin, kadmiumin ja vastaavien, seka N,N'-dibentsyylietyleenidiamiinista tai etyleenidiamiinista 15 muodostuneen orgaanisen kationin kanssa muodostetut emas-additiosuolat; ja (c) yhdistelmat, jotka koostuvat (a):sta ja (b):sta, esimerkiksi sinkkitannaattisuola ja vastaavat.Non-nicotinyl-D-lysyl Lys (Nic) Νε- (3-pyridyl) acetyl-D-lysyl Lys (pyridylacetyl) 30 3- (2,4,6-trimethylphenyl) alanyl Tmp 2,2-diphenylglycyl As used in the Dpg TS, the term "physiologically acceptable salts" means salts which retain the desired biological activity of the parent compound and which do not have any undesirable toxicological effects. Examples of such salts include 9 2326 δ (a) inorganic acids, e.g. acid addition salts formed with hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like, as well as organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, fumaric acid, fumaric acid, salts formed with as-corbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acids, naphthalenedisulfonic acids and polygalacturonic acid; base addition salts formed with metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium and the like, and an organic cation formed from N, N'-dibenzylethylenediamine or ethylenediamine; and (c) combinations consisting of (a) and (b), for example, zinc tannate salt and the like.
Ilmaisu "alempi alkyyli" tarkoittaa suoraketjuista 20 tai haaroittunutta, tyydytettya hiilivetyryhmaa, joka si-saitaa 1-4 hiiliatomia, kuten esimerkiksi metyylia, etyy-lia, n-propyylia, isopropyylia, n-butyylia, isobutyylia, s-butyylia ja t-butyylia. "1-6 hiiliatomia sisaitava al-kyyli" sulkee sisaansa samat substituentit kuin alempi 25 alkyyli mutta lisaksi mytts 5 tai 6 hiiliatomia sisaitavat ryhmat, kuten esimerkiksi n-pentyylin, n-heksyylin ja 5 tai 6 hiiliatomia sisaitavat haaroittuneet ryhmat. "1-12 hiiliatomia sisaitava alkyyli" sulkee sisaansa hiilivety-ryhmat, joissa on 1-12 hiiliatomia, mm. edelia mainitut 30 ryhmat silia erotuksella, etta ryhmat voivat sisaitaa jo-pa 12 hiiliatomia.The term "lower alkyl" means a straight or branched chain saturated hydrocarbon group containing from 1 to 4 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl and t-butyl. . "Alkyl having 1 to 6 carbon atoms" includes the same substituents as lower alkyl but additionally contains groups having 5 or 6 carbon atoms, such as, for example, n-pentyl, n-hexyl and branched groups having 5 or 6 carbon atoms. "Alkyl having 1 to 12 carbon atoms" includes hydrocarbon groups having 1 to 12 carbon atoms, e.g. the aforementioned 30 groups Silia with the difference that the groups can contain as many as 12 carbon atoms.
"Fluorialkyyli" tarkoittaa 1-5 fluoriatomilla substituoitua alempaa alkyylia, esimerkiksi ryhmia CF3CH2-, CFg-, CF3CF2CH2- ja vastaavia."Fluoroalkyl" means lower alkyl substituted with 1 to 5 fluorine atoms, for example, CF 3 CH 2, CF 3, CF 3 CF 2 CH 2 and the like.
35 "Halogeeni" tarkoittaa fluoria, klooria tai bro- mia.35 "Halogen" means fluorine, chlorine or bromine.
Lyhenne "N-Ac" tarkoittaa N-asetyyli-suojaryhmaa, so. terminaalisen aminohappotahteen amiinityppeen sitou- 92326 9 tunutta asetyyliryhmaa, yleisesti hyvåksytyn nimeåmista-van mukaisesti.The abbreviation "N-Ac" means an N-acetyl protecting group, i. an acetyl group bound to the amine nitrogen of the terminal amino acid residue, according to a generally accepted designation.
Edullisia tårnSn keksinndn mukaisia yhdisteita ovat yhdisteet, joissa 5 A on N-Ac-D-Nal(2); B on D-pF-Phe tal D-pCl-Phe; C on D-Trp tai D-Pal(3); D on Pal(2), Tyr, Arg, Deh, Mbh, Bth tai Pha; E on D-Trp, D-Tyr, D-Pal(3), D-Deh, D-Mbh, D-Bth tai 10 D-Pha; F on Leu tai Phe; G on Deh, Bth, Mbh tai Bha; ja J on D-AlaNl·^ tai GlyNl^.Preferred compounds of the invention are those wherein A is N-Ac-D-Nal (2); B is D-pF-Phe to D-pCl-Phe; C is D-Trp or D-Pal (3); D is Pal (2), Tyr, Arg, Deh, Mbh, Bth or Pha; E is D-Trp, D-Tyr, D-Pal (3), D-Deh, D-Mbh, D-Bth or D-Pha; F is Leu or Phe; G is Deh, Bth, Mbh or Bha; and J is D-AlaNl · or GlyN1 ^.
Viela edullisempia substituutiomalleja ovat sel-15 laiset, joissa A on N-Ac-D-Nal(2); B on D-pCl-Phe; C on D-Trp tai D-Pal(3); D on Tyr, Arg, Deh, Mbh, Bth tai Pha; 20 E on D-Trp, D-Pal(3), D-Tyr, D-Deh, D-Mbh, D-Bth tai D-Pha; F on Leu; G on Deh, Mbh, Bth tai Pha; ja J on D-AlaNH2.Even more preferred substitution patterns are those wherein A is N-Ac-D-Nal (2); B is D-pCl-Phe; C is D-Trp or D-Pal (3); D is Tyr, Arg, Deh, Mbh, Bth or Pha; E is D-Trp, D-Pal (3), D-Tyr, D-Deh, D-Mbh, D-Bth or D-Pha; F is Leu; G is Deh, Mbh, Bth or Pha; and J is D-AlaNH2.
- - 25 TSh3n edullisempaan ryhmaan kuuluu kolme edullista • · alaryhmaa: 1. Kun D on Tyr, E voi olla joko (a) jokin hydrofobisis-ta ryhmista D-Trp, D-Pal(3), ja D-Tyr, mutta edullisimmin D-Trp tai D-Pal(3); tai (b) jokin ryhmista D-Deh, D-Mbh, 30 D-Bth ja D-Pha.A more preferred group of 25 TSh 3 includes three preferred subgroups: 1. When D is Tyr, E may be either (a) one of the hydrophobic groups D-Trp, D-Pal (3), and D-Tyr, but most preferably D-Trp or D-Pal (3); or (b) any of D-Deh, D-Mbh, D-Bth and D-Pha.
2. Kun D on Arg, E on jokin kohdassa 1(a) luetelluista hydrofobisista ryhmista, edullisesti D-Tyr.2. When D is Arg, E is one of the hydrophobic groups listed in 1 (a), preferably D-Tyr.
3. Kun D on Deh, Mbh, Bth tai Pha, E on edullisimmin D-Tyr tai D-Pal(3), mutta mytts D-Trp on edullinen.3. When D is Deh, Mbh, Bth or Pha, E is most preferably D-Tyr or D-Pal (3), but mytts D-Trp is preferred.
35 Kussakin edullisista alaryhmista G on Deh, Mbh,35 In each of the preferred subgroups G is Deh, Mbh,
Bth tai Pha. Erityisen edullisia ovat Bth ja Deh ja edul- . lisin Deh.Bth or Pha. Particularly preferred are Bth and Deh and preferred. lisin Deh.
> · 92326 10> · 92326 10
Esimerkkejå edullisemmista substituutiomalleista ovat siis N-Ac-D-Nal(2)-D-pCl-Phe-C-Ser-Tyr-C-Leu-G-Pro-D-AlaNI^, jossa C on D-Trp tai D-Pal(3) ja G on Deh, Mbh, Bth tai Pha; N-Ac-D-Nal(2)-D-pCl-Phe-C-Ser-Tyr-E-Leu-G-5 Pro-D-AlaNH2, jossa C on D-Trp tai D-Pal(3), E on D-Deh, D-Bth, D-Mbh tai D-Pha ja G on tåssS kappaleessa esite-tyn mååritelmån mukaisen E:n L-muoto; N-Ac-D-Nal(2)-D-pCl-Phe-C-Ser-Arg-E-Leu-G-Pro-D-AlaNH2, jossa C on D-Trp tai D-Pal(3), E on D-Trp, D-Pal(3) tai D-Tyr ja G on Deh, 10 Mbh, Bth tai Pha; ja N-Ac-D-Nal(2)-D-pCl-Phe-C-Ser-D-E-Thus, examples of more preferred substitution patterns are N-Ac-D-Nal (2) -D-pCl-Phe-C-Ser-Tyr-C-Leu-G-Pro-D-AlaNI, where C is D-Trp or D- Pal (3) and G are Deh, Mbh, Bth or Pha; N-Ac-D-Nal (2) -D-pCl-Phe-C-Ser-Tyr-E-Leu-G-5 Pro-D-AlaNH2, where C is D-Trp or D-Pal (3), E is D-Deh, D-Bth, D-Mbh or D-Pha and G is the L-form of E as defined in this paragraph; N-Ac-D-Nal (2) -D-pCl-Phe-C-Ser-Arg-E-Leu-G-Pro-D-AlaNH2, where C is D-Trp or D-Pal (3), E is D-Trp, D-Pal (3) or D-Tyr and G is Deh, 10 Mbh, Bth or Pha; and N-Ac-D-Nal (2) -D-pCl-Phe-C-Ser-D-E-
Leu-G-Pro-D-AlaNH2, jossa C on D-Trp tai D-Pal(3), D ja G ovat kumpikin itsenåisesti Deh, Bth, Mbn tai Pha ja E on D-Tyr, D-Trp tai D-Pal(3).Leu-G-Pro-D-AlaNH2, where C is D-Trp or D-Pal (3), D and G are each independently Deh, Bth, Mbn or Pha and E is D-Tyr, D-Trp or D- Pal (3).
Muita edullisia toteutusmuotoja ovat 15 N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-E-Leu-G-Pro-D-Ala- NH2, jossa E on D-Trp tai D-Tyr ja G on Deh, Bth, Mbh tai Pha; N-Ac-D-Nal(2)-D-pCl-Phe-C-Ser-D-E-Leu-G-Pro-D-AlaNH2, jossa D ja G ovat kumpikin itsenåisesti Deh, Bth, Mbh tai 20 Pha ja C ja E ovat kumpikin itsenåisesti D-Trp tai D-Pal(3); ja N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Pal(3)-Leu-G-Pro-D-AlaNH2, jossa G on Deh, Bth, Mbh tai Pha.Other preferred embodiments are N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Tyr-E-Leu-G-Pro-D-Ala-NH2, where E is D-Trp or D-Tyr and G is Deh, Bth, Mbh or Pha; N-Ac-D-Nal (2) -D-pCl-Phe-C-Ser-DE-Leu-G-Pro-D-AlaNH2, where D and G are each independently Deh, Bth, Mbh or 20 Pha and C and E is each independently D-Trp or D-Pal (3); and N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Tyr-D-Pal (3) -Leu-G-Pro-D-AlaNH2, where G is Deh , Bth, Mbh or Pha.
Esimerkkejå edullisista toteutusmuodoista ovat -25 seuraavat: • · N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Pal(3)-Leu-Deh-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Pal(3)-Leu-Bth-Pro-D-AlaNH2; 30 N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Pal(3)-Examples of preferred embodiments are -25 as follows: • N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Tyr-D-Pal (3) -Leu-Deh-Pro -D-AlaNH2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Tyr-D-Pal (3) -Leu-Bth-Pro-D-AlaNH 2; 30 N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Tyr-D-Pal (3) -
Leu-Mbh-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Pal(3)-Leu-Pha-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Trp-Leu-35 Deh-Pro-D-AlaNI^; N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Trp-Leu-Leu-Mbh-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Tyr-D-Pal (3) -Leu-Pha-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Tyr-D-Trp-Leu-35 Deh-Pro-D-AlaNI ^; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Tyr-D-Trp-Leu-
Bth-Pro-D-AlaNH0; • z 11 52326 N-Ac-D-Nal(2)-D-pCl-Phs-D-Trp-Ser-Tyr-D-Trp-Leu-Mbh-Pro-D-AlaNH2; U_Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Trp-Bth-Pro-D-AlaNH0; • z 11 52326 N-Ac-D-Nal (2) -D-pCl-Phs-D-Trp-Ser-Tyr-D-Trp-Leu-Mbh-Pro-D-AlaNH2; U_Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Tyr-D-Trp
Leu-Pha-Pro-D-AlaNH2; 5 N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Deh-Leu-Pha-Pro-D-AlaNH 2; 5 N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Tyr-D-Deh-
Leu-Deh-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Mbh-Leu-MPh-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Bth-10 Leu-Bth-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Pna-Leu-Pha-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Deh-Leu-Deh-Pro-D-AlaNH2; ^ N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Mbn-Leu-Deh-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Tyr-D-Mbh-Leu-MPH-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Tyr-D-Bth-10 Leu-Bth-Pro-D-AlaNH2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Tyr-D-PNA-Leu-Pha-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Tyr-D-Deh-Leu-Deh-Pro-D-AlaNH 2; ^ N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Tyr-D-Mbn-
Leu-Mdh-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-3th-Leu-Bth-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Pha-20 Leu-Pha-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Arg-D-Trp-Leu-Deh-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Arg-D-Trp-Leu-Bth-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Arg-D-Trp-Leu-Mbh-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Arg-D-Trp-Leu-Pha-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Arg-D-Pal(3) -Leu-Bth-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Arg-D-Pal(3)-Leu-Mbh-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Arg-D-Pal(3) -MDH-Leu-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Tyr-D-3th-Leu-Bth-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Tyr-D-Pha-20 Leu-Pha-Pro-D-AlaNH2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Arg-D-Trp-Leu-Deh-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Arg-D-Trp-Leu-Bth-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Arg-D-Trp-Leu-Mbh-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Arg-D-Trp-Leu-Pha-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Arg-D-Pal (3) -Leu-Bth-Pro-D-AlaNH2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Arg-D-Pal (3) -Leu-Mbh-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Arg-D-Pal (3) -
Leu-Deh-Pro-D-AlaNH2; 35 12 92326 N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Arg-D-Pal(3)-Leu-Deh-Pro-D-AlaNH 2; 35 12 92326 N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Arg-D-Pal (3) -
Leu-Pha-Pro-D-AlaNH2; N_Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Arg-D-Tyr-Leu-Den-Pro-D-AlaNH2; 5 N-Ac-D-Nal(2)-D-pCl-Phe-D-T rp-Ser-Arg-D-Tyr-Leu-Pha-Pro-D-AlaNH 2; N_Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Arg-D-Tyr-Leu-Den-Pro-D-AlaNH 2; 5 N-Ac-D-Nal (2) -D-pCl-Phe-D-T rp-Ser-Arg-D-Tyr-
Leu-Bth-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Arg-D-Tyr-Leu-MDh-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Arg-D-TyΓΙΟ Leu-Pha-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Arg-D-Tyr-Leu-Bth-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Arg-D-Tyr-Leu-MDH-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Arg-D-TyΓΙΟ Leu-Pha-Pro-D-AlaNH2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Arg-D-Tyr
Leu-Bth-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Arg-D-Tyr-Leu-Mbh-Pro-D-AlaNH2; 15 N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Arg-D-Tyr-Leu-Bth-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Arg-D-Tyr-Leu-Mbh-Pro-D-AlaNH 2; 15 N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Arg-D-Tyr-
Leu-Deh-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Arg-D-Tyr-Leu-Pha-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-T rp-Ser-Deh-D-Tyr-Leu-20 Deh-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Mbh-D-Tyr-Leu-Mbh-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Bth-D-Tyr-Leu-Bth-Pro-D-AlaNH2; 25 N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Pha-D-Tyr-Leu-Leu-Deh-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Arg-D-Tyr-Leu-Pha-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-T rp-Ser-Deh-D-Tyr-Leu-20 Deh-Pro-D-AlaNH2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Mbh-D-Tyr-Leu-Mbh-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Bth-D-Tyr-Leu-Bth-Pro-D-AlaNH 2; 25 N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Pha-D-Tyr-Leu-
Pha-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Deh-D-T rp-Leu-Deh-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Mbh-D-Trp-Leu-30 Mbh-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Bth-D-Trp-Leu-Bth-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Pha-D-Trp-Leu-Pha-Pro-D-AlaNH2; 35 13 92326 N-Ac-D-Nal(2)-D-pCl-Phs-D-Pal(3)-Ser-Deh-D-Pal(3)-Le'J-Deh-Pro-D-AlaNh2; N-Ac-D-Nal(2)-D-pCl-Pne-D-Pal(3)-Ser-Mbn-D-Pal(3)-Leu-MDh-Pro-U-AlaNH2; 5 N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-3th-D-Pal(3)-Pha-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Deh-D-T rp-Leu-Deh-Pro-D-AlaNH2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Mbh-D-Trp-Leu-30 Mbh-Pro-D-AlaNH2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Bth-D-Trp-Leu-Bth-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Pha-D-Trp-Leu-Pha-Pro-D-AlaNH 2; 35 13 92326 N-Ac-D-Nal (2) -D-pCl-Phs-D-Pal (3) -Ser-Deh-D-Pal (3) -Le'J-Deh-Pro-D-AlaNh2; N-Ac-D-Nal (2) -D-pCl-pne-D-Pal (3) -Ser-MBN D-Pal (3) -Leu-Pro-MDH-U-AlaNH 2; 5 N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-3th-D-Pal (3) -
Leu-Bth-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Pha-D-Pal(3)-Leu-Pha-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Deh-D-Nal(2)-10 Leu-Deh-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Mbh-D-Nal(2)-Leu-Mbh-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-T rp-Ser-3th-D-Nal(2) -Leu-Bth-Pro-D-AlaNH2; 15 N-Ac-D-Nal(2)-D-pCl-Phe-D-T rp-Ser-Pha-D-Nal(2)-Leu-Bth-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Pha-D-Pal (3) -Leu-Pha-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Deh-D-Nal (2) -10 Leu-Deh-Pro-D-AlaNH2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Mbh-D-Nal (2) -Leu-Mbh-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-T rp-Ser-3th-D-Nal (2) -Leu-Bth-Pro-D-AlaNH2; 15 N-Ac-D-Nal (2) -D-pCl-Phe-D-T rp-Ser-Pha-D-Nal (2) -
Leu-Pha-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Pal(3)-D-Pal(3)-Leu-Deh-Pro-D-AlaNH2; .Leu-Pha-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Pal (3) -D-Pal (3) -Leu-Deh-Pro-D-AlaNH 2; .
N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Pal(3)-20 D-Pal(3)-Leu-Bth-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Pal(3)- D-Pal(3)-Leu-Pha-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Pal(3)- D-Pal(3)-Leu-Mbh-Pro-D-AlaNH2; 25 N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Mbh-Leu- 3tn-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Mbh-Leu-Bth-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pF-Phe-D-Pal(3)-Ser-Tyr-D-Pal(3)- 30 Leu-Deh-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pF-Phe-D-Pal(3)-Ser-Tyr-D-Pal(3) -N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Pal (3) -20 D-Pal (3) -Leu-Bth-Pro-D-AlaNH2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Pal (3) -D-Pal (3) -Leu-Pha-Pro-D-AlaNH2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Pal (3) -D-Pal (3) -Leu-Mbh-Pro-D-AlaNH2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Tyr-D-Mbh-Leu- 3tn-Pro-D-AlaNH2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Tyr-D-Mbh-Leu-Bth-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pF-Phe-D-Pal (3) -Ser-Tyr-D-Pal (3) -30 Leu-Deh-Pro-D-AlaNH2; N-Ac-D-Nal (2) -D-pF-Phe-D-Pal (3) -Ser-Tyr-D-Pal (3) -
Leu-Bth-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pF-Pl^e-D-Trp-SeΓ-Tyr-D-TΓp-Leu-Bth-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pF-PI ^ E-D-Trp-SeΓ-Tyr-D-TΓp-
Leu-Deh-Pro-D-AlaNH2; -35 92326 14 N-Ac-D-Nal(2)-D-pF-Phe-D-Trp-Ser-Tyr-D-Trp-Leu-Deh-Pro-D-AlaNH 2; -35 92326 14 N-Ac-D-Nal (2) -D-pF-Phe-D-Trp-Ser-Tyr-D-Trp-
Leu-3th-Pro-D-rtlaNH2; N-Ac-D-Nal(2)-D-pF-Phe-D-Trp-Ser-Tyr-D-Den-3th-Leu-Pro-D-rtlaNH2; N-Ac-D-Nal (2) -D-pF-Phe-D-Trp-Ser-Tyr-D-Denhardt
Leu-Deh-Pro-D-AlaNH2; 5 N-Ac-D-Nal(2)-D-pF-Phe-D-T rp-Ser-Tyr-D-Bth-Leu-Deh-Pro-D-AlaNH 2; 5 N-Ac-D-Nal (2) -D-pF-Phe-D-T rp-Ser-Tyr-D-Bth-
Leu-Bth-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pF-Phe-D-Pal(3)-Ser-Tyr-D-Den-Leu-Deh-Pro-D-AlaNH^; N-Ac-D-Nal(2)-D-pF-Phe-D-Pal(3)-Ser-Tyr-D-Bth-10 Leu-3th-Pro-D-AlaNH2; N-Ac-D-Nal (2)-D-pF-Phe-D-Trp-Ser-Arg-D-T rp-Leu-Deh-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pF-Phe-D-Trp-Ser-Arg-D-Trp-Leu-3th-Pro-D-AlaNH2; 15 N-Ac-D-Nal(2)-D-pF-Phe-D-Pal(3)-Ser-Arg-D-Pal(3)-Leu-Bth-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pF-Phe-D-Pal (3) -Ser-Tyr-D-Den-Leu-Deh-Pro-D-AlaNH ^; N-Ac-D-Nal (2) -D-pF-Phe-D-Pal (3) -Ser-Tyr-D-Bth-10 Leu-3th-Pro-D-AlaNH2; N-Ac-D-Nal (2) -D-pF-Phe-D-Trp-Ser-Arg-D-T rp-Leu-Deh-Pro-D-AlaNH2; N-Ac-D-Nal (2) -D-pF-Phe-D-Trp-Ser-Arg-D-Trp-Leu-3th-Pro-D-AlaNH 2; 15 N-Ac-D-Nal (2) -D-pF-Phe-D-Pal (3) -Ser-Arg-D-Pal (3) -
Leu-3th-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pF-Phe-D-Pal(3)-Ser-Arg-D-Pal(3)-Leu-Deh-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pF-Phe-D-T rp-Ser-Deh-D-Tyr-Leu-20 Deh-Pro-D-AlaNH2; and N-Ac-D-Nal(2)-D-pF-Phe-D-Trp-Ser-Bth-D-Tyr-Leu-Bth-Pro-D-AlaNH2.3th-Leu-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pF-Phe-D-Pal (3) -Ser-Arg-D-Pal (3) -Leu-Deh-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pF-Phe-D-T rp-Ser-Deh-D-Tyr-Leu-20 Deh-Pro-D-AlaNH2; and N-Ac-D-Nal (2) -D-pF-Phe-D-Trp-Ser-Bth-D-Tyr-Leu-Bth-Pro-D-AlaNH2.
Tåman keksinnon piiriin kuuluu myos peptideja,jotka eivat valttamatta kuulu edella mainittuihin edullisiin ryftniin, kuten 25 esimerkiksi seuraavat: N-Ac-D-Nal(2)-D-pC1-Phe-D-T rp-Ser-Tyr-D-Tyr-Leu-Deh-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Tyr-Leu-Mbh-Pro-D-AlaNH2; 30 N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Tyr-Also included within the scope of this invention are peptides that are not necessarily included in the aforementioned preferred compounds, such as the following: N-Ac-D-Nal (2) -D-pC1-Phe-DT rp-Ser-Tyr-D-Tyr-Leu -Deh-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Tyr-D-Tyr-Leu-Mbh-Pro-D-AlaNH 2; 30 N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Tyr-D-Tyr-
Leu-Bth-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Tyr-Leu-Pha-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-35 D-Nal(2)-Leu-Deh-Pro-D-AlaNH2; « · > • » 92326 15 N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Nal(2) -Leu-Mbh-Pro-D-AlaNI^; N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Nal(2) -Leu-Bth-Pro-D-AlaNI^ · 5 N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Na-(2) -Leu-Bth-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Tyr-D-Tyr-Leu-Pha-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Tyr-35 D-Nal (2) -Leu-Deh-Pro-D-AlaNH2; «·> •» 92326 15 N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Tyr-D-Nal (2) -Leu-Mbh-Pro-D- Alani ^; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Tyr-D-Nal (2) -Leu-Bth-Pro-D-AlaNI ^ · 5 N-Ac -D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Tyr-D-Na- (2) -
Leu-Pha-Pro-D-AlaNI^; N-Ac-D-Nal(2)-D-aMe,pCl-Phe-D-Pal(3)-Ser-Arg-D-Pal(3)-Leu-Btn-Pro-D-AlaNI^; N-Ac-D-Nal(2)-D-aMe,pCl-Phe-D-Pal(3)-Ser-Tyr-D-10 Pal(3)-Leu-Bth-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-aMe,pCl-Phe-D-Trp-Ser-Arg-D-Trp-Leu-Bth-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Glu(AA)-Leu-Bth-Pro-D-AlaNH2; 15 N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Arg-D-Leu-Pha-Pro-D-Alani ^; N-Ac-D-Nal (2) -D-AME, pCl-Phe-D-Pal (3) -Ser-Arg-D-Pal (3) -Leu-Btn-Pro-D-Alani ^; N-Ac-D-Nal (2) -D-aMe, pCl-Phe-D-Pal (3) -Ser-Tyr-D-10 Pal (3) -Leu-Bth-Pro-D-AlaNH2; N-Ac-D-Nal (2) -D-AME, pCl-Phe-D-Trp-Ser-Arg-D-Trp-Leu-Bth-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Tyr-D-Glu (AA) -Leu-Bth-Pro-D-AlaNH 2; 15 N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Arg-D-
Glu(AA)-Leu-Bth-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Arg-D-Glu(AA)-Leu-Bth-Pro-D-AlaNH2 ? N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-20 Pal(3)-Leu-HArg(CH2CF2CF3)-Pro-D-AlaNH2; ja N-Ac-D-Nal(2)-Dpg-D-Pal(3)-Ser-Tyr-D-Pal(3)-Nal(2)-Bth-Pro-D-AlaNH2.Glu (AA) -Leu-Bth-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Arg-D-Glu (AA) -Leu-Bth-Pro-D-AlaNH2? N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Tyr-D-20 Pal (3) -Leu-HArg (CH2CF2CF3) -Pro-D-AlaNH2; and N-Ac-D-Nal (2) -Dpg-D-Pal (3) -Ser-Tyr-D-Pal (3) -Nal (2) -Bth-Pro-D-AlaNH2.
On yleenså edullista, etta A-, B-, C-, E- ja J-aminohappotahde ovat D-isomeerin muodossa ja etta D-, F-.25 ja G-amlnohappotahde ovat L-isomeerin muodossa. Stereoke- • · mia tulee kaslttaa tailalseksl, ellei sita ole toisenlai-seksl erikseen malnlttu.It is generally preferred that the A, B, C, E and J amino acid residues be in the form of the D-isomer and that the D, F-.25 and G-amino acid residues be in the form of the L-isomer. Stereochemistry should be used unless otherwise specified.
Kaiklssa edelia mainituissa toteutusmuodoissa yh-dlste voidaan valmlstaa myds vastaavan farmaseuttisesti 30 hyvaksyttavan suolan muodossa.In all of the foregoing embodiments, the compound may be prepared in the form of a corresponding pharmaceutically acceptable salt.
Tdman kekslnnOn mukaisilla yhdlsteilia ja erityi-sestl nliden suolollla on yliattavan volmakas ja pltkaan kestava LHRH-antagonlstlnen aktllvlsuus.The compounds of the present invention, and in particular the salts thereof, have an excessively potent and long-lasting LHRH antagonist activity.
1616
9.1 o L O9.1 o L O
LHRH-antagonismin voimakkuuden ensisijainen mitta on kyky estaa ovulaatiota rotilla, mika maaritetaan A. Corbinin ja C.W. Beattien menetelmaiia (Endocrine Res. Commun. 2 (1975) 1).The primary measure of the potency of LHRH antagonism is the ability to inhibit ovulation in rats, as determined by A. Corbin and C.W. The methods of the Beatts (Endocrine Res. Commun. 2 (1975) 1).
5 Kyky saada aikaan histamiinin vapautuminen rotan peritoneaalisista syttttttsoluista in vitro voidaan mSSrit-taa tavalla, jonka ovat esittaneet Sydbom ja Terenius (Agents and Action 16 (1985) 269) tai Siraganian et al. (Manual of Clinical Immunology, 2. painos, toim. N.E.The ability to induce histamine release from rat peritoneal stem cells in vitro can be mSSrit as described by Sydbom and Terenius (Agents and Action 16 (1985) 269) or Siraganian et al. (Manual of Clinical Immunology, 2nd ed., Ed. N.E.
10 Rose ja M. Friedman, Amer. Soc. Microbiol., Washington, D.C., 1980, s. 808).10 Rose and M. Friedman, Amer. Soc. Microbiol., Washington, D.C., 1980, p. 808).
Muita biologisia testeja, joita voidaan kayttaa LHRH:n antagonistien ja taman keksinnttn mukaisten yhdis-teiden tutkimiseen, ovat 15 (a) LHRH:n aiheuttaman FSH:n ja LH:n vapautumisen inhi- bitio rotalla in vivo (J.A. Vilchez-Martinez et al., Endocrinology 96 (1975) 1130); (b) aivolisakkeen etulohkon solujen dispersioviljelman aikaansaama LH:n ja FSH:n vapautumisen inhibitio radio- 20 immunologisesti mitattuna (W. Vale et al., Endocrinology 91 (1972) 562); ja (c) gonadotropiinitasojen alentaminen kastroidulla rotalla ja koiralla (Petrie et al., Male Contraception, Harper and Row, Philadelphia, 1985, s. 361).Other biological assays that can be used to study LHRH antagonists and compounds of this invention include the inhibition of LHRH-induced FSH and LH release in rats in vivo (JA Vilchez-Martinez et al. ., Endocrinology 96 (1975) 1130); (b) inhibition of LH and FSH release by radioimmunological measurement by dispersion culture of anterior pituitary cells (W. Vale et al., Endocrinology 91 (1972) 562); and (c) lowering gonadotropin levels in castrated rats and dogs (Petrie et al., Male Contraception, Harper and Row, Philadelphia, 1985, p. 361).
25 Antagonistiset vaikutukset ja kayttttmahdollisuudet • é25 Antagonistic effects and potential uses • é
Tassa kuvattujen yhdisteiden antagonistisesta vai-kutuksesta johtuvat seuraavat kayttttmahdollisuudet: - hedelmttityksen ehkaisy naarailla, - ovulaation esto tai viivytys, 30 - hedelmttityksen ehkaisy uroksilla, - diagnostiset kayttttmahdollisuudet, kuten osteoporoosin diagnosointi, - muut kayttttmahdollisuudet, joita on esitetty artikke-lissa B. H. Vickery, Endocrine Reviews 7 (1986) 115.The following uses result from the antagonistic effect of the compounds described here: - prevention of infertility in females, - prevention or delay of ovulation, 30 - prevention of infertility in males, - diagnostic uses such as the diagnosis of osteoporosis, - other uses described in B Reviews 7 (1986) 115.
IIII
17 9232617 92326
Sovellettaessa taman keksinndn mukaisia yhdisteitS esim. ovulaation estoon halutulle kohteelle annetaan vai-kuttava maarS taman keksinnOn mukaista yhdistetta tai si-ta slsaitavaa koostumusta. Nama yhdisteet tai koostumuk-5 set voidaan antaa eri tavoilla sen mukaan, mika on tarkka kaytttttarkoitus, mm. suun kautta, parenteraalisesti (mika kasittaa ihonalaisen, lihaksensisaisen ja laskimonsisai-sen annon), emattimen kautta (erityisesti hedelmttityksen ehkaisyn tapauksessa), perasuolen kautta, bukkaalisesti 10 (kuten esimerkiksi kielen alle), ihon lapi tai nenan kautta. Sopivin tie kussakin eri tapauksessa riippuu kayttdtarkoituksesta, kulloisestakin vaikuttavasta aine-osasta ja kohteesta. Yhdiste tai koostumus voidaan antaa myiSs saannttstellysti kayttaen vaikuttavaa aineosaa va-15 pauttavia depotistute- tai ruiskeformuloita, joita kuva-taan tarkemmin jaljempana.When applying the compounds of this invention, e.g. to inhibit ovulation, the desired subject is administered a compound of the present invention or a composition therefor. These compounds or compositions may be administered in various ways depending on the precise use, e.g. via the oral, parenteral (which includes subcutaneous, intramuscular and its laskimonsisai-administration), vaginally (particularly for hedelmttityksen prevention), rectally, buccally 10 (such as, for example, sublingual), transdermal or intranasal. The most appropriate route in each case depends on the intended use, the active ingredient and the target. The compound or composition may also be administered in a controlled manner using active ingredient depot or injection formulations, which are described in more detail below.
Mita edelia esitettyihin kayttOtarkoituksiin tu-lee, vaikuttavaa aineosaa on yleensa tarkoituksenmukaista antaa noin 0,001 - 5 mg/ruumiinpaino-kg. Ihmisille ainet-20 ta annetaan edullisesti noin 0,01-1 mg/kg/vuorokausi ja eiaimille noin 0,1-1 mg/kg/vuorokausi. Tama antaminen voi tapahtua yhdelia kertaa, jakamalla annos useaan antoeraan tai vaikuttavaa aineosaa hitaasti vapauttavassa muodossa tehokkaimpien tulosten saavuttamiseksi. Kun kysymyksessa 25 on kiiman tai tiineyden ehkaisy eiaimilia, annos on edul- • · lisimmin noin 1-10 mg/kg ja se annetaan yhtena annoksena.For the foregoing uses, it will generally be expedient to administer the active ingredient in an amount of from about 0.001 to about 5 mg / kg of body weight. The substances are preferably administered to humans at about 0.01-1 mg / kg / day and to non-humans at about 0.1-1 mg / kg / day. This administration may be performed in a single dose, in divided doses, or in a sustained release form for the most effective results. In the case of question 25 for the prevention of heat or pregnancy, the dose is most preferably about 1 to 10 mg / kg and is administered in a single dose.
Parenteraalinen anto edellyttaa yleensa pienempaa annostusta kuin muut antornenetelmat, jotka ovat enemman imeytymisesta riippuvaisia.Parenteral administration generally requires a lower dosage than other methods of administration that are more dependent on absorption.
30 KeksintO koskee myOs koostumuksia, jotka sisalta- vat vaikuttavana aineosana taman keksinnOn mukaista yh- ♦ distetta, joka on sekoitettu johonkin fysiologisesti hy-vaksyttavaan myrkyttomaan kantajaan. Kuten edelia on mai-nittu, sellaiset koostumukset voidaan valmistaa kaytetta- I · 18 92326 viksi parenteraaliseen (ihonalaiseen, lihaksensisaiseen tai laskimonsisåiseen) antamiseen erityisesti nestemais-ten liuosten ja suspensioiden muodossa; kaytettaviksi emattimen tai perasuolen kautta antamiseen erityisesti 5 puolikiinteissa muodoissa, kuten esimerkiksi voiteina ja perapuikkoina; kaytettaviksi suun kautta tai bukkaalises-ti antamiseen erityisesti tablettien tai kapselien muodossa; tai kaytettaviksi nenan kautta erityisesti pulve-reiden, nenatippojen tai aerosolien muodossa.The invention also relates to compositions comprising as active ingredient a compound of this invention ♦ in admixture with a physiologically acceptable non-toxic carrier. As mentioned above, such compositions may be formulated for parenteral (subcutaneous, intramuscular or intravenous) administration, especially in the form of liquid solutions and suspensions; for use in vaginal or rectal administration particularly in semisolid forms 5, such as creams and suppositories; for oral or buccal administration, especially in the form of tablets or capsules; or for nasal administration, in particular in the form of powders, nasal drops or aerosols.
10 Koostumukset voidaan antaa katevasti yksikkOannos- muodossa ja voidaan valmistaa milla tahansa niista mene-telmista, jotka ovat farmasian alalla tunnettuja ja joi-ta esimerkiksi kuvataan teoksessa Remington’s Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsyl-15 vania, 1970. Parenteraalisesti annettaviksi tarkoitetut formulat voivat sisaitaa tavanomaisia tSyteaineita, esimerkiksi steriilia vetta tai fysiologista suolaliuosta, alkyleeniglykoleja, kuten propyleeniglykolia, polyalky-leeniglykoleja, kuten polyetyleeniglykolia, kasviperaisia 20 dljyja, hydrattuja naftaleeneja seka vastaavia. Emattimen tai perasuolen kautta annettaviksi tarkoitetut formulat, esimerkiksi perapuikot, voivat sisaitaa tayteaineina esimerkiksi polyalkyleeniglykoleja, vaseliinia, kaakaovoita ja vastaavia. Nenan kautta annettaviksi tarkoitetut for-25 mulat voivat olla kiinteita ja sisaitaa tayteaineina esi- · merkiksi laktoosia ja dekstraania tai olla vesi- tai 01-jyliuoksia, jotka on tarkoitettu annettaviksi nenatippojen tai annostellun suihkeen muodossa. Bukkaalisissa an-tomuodoissa tyypillisia tayteaineita ovat sokerit, kal-30 siumstearaatti, magnesiumstearaatti, esigelatinoitu tark-kelys ja vastaavat.The compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsyl-15, 1970. For parenteral administration. the formulations may contain conventional cytotoxic agents, for example, sterile water or physiological saline, alkylene glycols such as propylene glycol, polyalkylene glycols such as polyethylene glycol, vegetable oils, hydrogenated naphthalenes and the like. The formulations intended for administration via the vaginal or rectal use, for example, suppositories, may your built-fillers, for example, polyalkylene glycols, petroleum jelly, cocoa butter and the like. Formulations for nasal administration may be solid and may contain as excipients, for example, lactose and dextran, or may be aqueous or non-aqueous solutions intended for administration in the form of nasal drops or metered spray. Typical excipients for buccal administration include sugars, calcium stearate, magnesium stearate, pregelatinized observation, and the like.
Taman keksinnbn mukaisten nona- ja dekapeptidien antaminen nenan kautta on erityisen edullista. Imeytymis-ta nenan limakalvon lapi parantavat pinta-aktiiviset ha- 19Nasal administration of the nona and decapeptides of this invention is particularly preferred. Absorption through the nasal mucosa is enhanced by surfactants
O o 7 O L "s lO LOO o 7 O L "s lO LO
pot, kuten esimerkiksi glykokoolihappo, koolihappo, tau-rokoolihappo, kolaanihappo, etokoollhappo, deoksikooli-happo, kenodeoksikoolihappo, dehydrokoolihappo ja glyko-deokslkoollhappo.pot such as glycocholic acid, cholic acid, tau rocholic acid, cholanoic acid, etocholic acid, deoxycholic acid, chenodeoxycholic acid, dehydrocolic acid and glyco deoxycholic acid.
5 LHRH:n antagonistia sisSltav&ån liuos- tai jauhe- formulaan voidaan lis&tS yhta tai useampaa pinta-aktii-vista happoa tai suolaa mutta edullisesti yhta fysiologisesti hyvaksyttavaa happosuolaa. Sopivia fysiologisesti hyvSksyttSvia pinta-aktiivisia suoloja ovat suolat, joita 10 kaytettaessa peptidin parantunut imeytyminen samoin kuin yhdisteen pinta-aktiiviset ominaisuudet sailyvat ja jotka eivåt ole kohteelle vahingollisia tai muista syista sovel tumattomia. Sellaisia suoloja ovat esimerkiksi epaor-gaanisista emaksista saatavat suolat, mm. natrium-, ka-15 lium-, litium-, ammonium-, kalsium-, magnesium-, alumii-ni-, rauta(III)- ja magnaani(III)suolat seka vastaavat. Erityisen edullisia ovat ammonium-, kalium-, natrium-, kalsium- ja magnesiumsuolat. Fysiologisesti hyvaksytta-vista myrkyttdmista orgaanisista emaksista saatavia suo-20 loja ovat primaaristen, sekundaaristen ja tertiaaristen amiinien, substituoitujen amiinien (luonnossa esiintyvdt substituoidut amiinit mukaan luettuina), syklisten amiinien ja emaksisten ioninvaihtohartsien, kuten esimerkiksi isopropyyliamiinin, trimetyyliamiinin, dietyyliamiinin, 25 trietyyliamiinin, tripropyyliamiinin, etanolamiinin, 2- I · dimetyyliaminoetanolin, 2-dietyyliaminoetanolin, tromet-amiinin, disykloheksyyliamiinin, lysiinin, arginiinin, histidiinin, kofeiinin, prokaiinin, hydrabamiinin, kolii-nin, betaiinin, etyleenidiamiinin, glykosamiinin, metyy-30 liglukamiinin, teobromiinin, puriinien, piperatsiinin, piperidiinin, N-etyylipiperidiinin, polyamiinihartsien seka mulden vastaavien, suolat. Erityisen edullisia myr-kyttOmia orgaanisia emaksia ovat isopropyyliamiini, di-etyyliamiini, etanolamiini, trometamiini, disykloheksyy-35 liamiini, koliini ja kofeiini.One or more surfactant acids or salts, but preferably one physiologically acceptable acid salt, may be added to the solution or powder formulation containing the LHRH antagonist. Suitable physiologically acceptable surfactant salts are those which, when used, preserve the improved absorption of the peptide as well as the surfactant properties of the compound and which are not harmful to the subject or otherwise unsuitable. Such salts include, for example, salts obtained from inorganic emulsions, e.g. sodium, ka-15 lithium, lithium, ammonium, calcium, magnesium, aluminum, iron (III) and magnesium (III) salts and the like. Ammonium, potassium, sodium, calcium and magnesium salts are particularly preferred. Salts from physiologically acceptable toxic organic bases include primary, secondary and tertiary amines, substituted amines (including naturally occurring substituted amines), tripropylamine, cyclopamine amines, cyclopamine amines, triethylamines, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, tromethamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylamine, ethylenediamine, glycosamine, methylamine, methylamine , salts of piperidine, N-ethylpiperidine, polyamine resins and the like. Particularly preferred toxic organic bases are isopropylamine, diethylamine, ethanolamine, tromethamine, dicyclohexylamine, choline and caffeine.
. · 20 9'/ ό i 6. · 20 9 '/ ό i 6
Vieia edullisemmin tSmSn keksinnOn kåytanniin so-vellutuksissa kaytettava pinta-aktiivinen aine on glyko-koolihapon alkalimetallisuola, edullisimmin natriumglyko-kolaatti.More preferably, the surfactant used in the practice of the present invention is an alkali metal salt of glycolic acid, most preferably sodium glycolate.
5 Pinta-aktiivista ainetta kaytetaan taman keksin nOn kaytanndn sovellutuksissa sellaisena maarana, ettå se parantaa LHRH-peptidien imeytymista enemman kuin muut pinta-aktiiviset aineet, jotka saattavat parantaa pepti-dien imeytymista jossakin maarin. On havaittu, etta usein 10 sellainen maara on 0,2-15 % (massa/tilavuus), vieia useammin 0,2-5 % (massa/tilavuus) liuoksesta. Pinta-aktiivista ainetta on edullista olla mukana noin 0,5-4 % (massa/tilavuus), mieleliaan noin 1 % (massa/tilavuus) ja edullisesti noin 2 % (massa/tilavuus).The surfactant is used in the applications of the present invention to the extent that it enhances the absorption of LHRH peptides more than other surfactants that may enhance the absorption of the peptides in any of the paints. It has been found that often such an amount is 0.2-15% (w / v), more often 0.2-5% (w / v) of solution. The surfactant is preferably present in an amount of about 0.5-4% (w / v), preferably about 1% (w / v) and preferably about 2% (w / v).
15 Naihin formuloihin voidaan lisata muitakin ainei- ta, kuten esimerkiksi sailymista parantavia aineita, suo-loja kudosta vastaavan elektrolyyttipitoisuuden saavutta-miseksi seka muita lisaaineita, jotka ovat nenan kautta annettaviksi tarkoitettujen formuloiden kemiassa tunnet-20 tuja. Erityisen edullisia sellaisia muita aineita ovat pinta-aktiiviset aineet, edullisesti ionittomat pinta-aktiiviset aineet, kuten polysorbaatit, 0,1-5 %:n (massa/tilavuus), vieia edullisemmin 0,25-2 %:n (massa/tilavuus ), pitoisuuksina.Other substances may be added to these formulations, such as preservatives, salts to achieve a tissue-like electrolyte content, and other additives known in the chemistry of nasal formulations. Particularly preferred such other substances are surfactants, preferably nonionic surfactants such as polysorbates, in an amount of 0.1-5% (w / v), more preferably 0.25-2% (w / v) , in concentrations.
25 On havaittu, etta paremman liukenevuuden ja sta- • 1 biilisuuden saavuttamiseksi sappihapon ja peptidin mooli-suhteen on tarkoituksenmukaista olla vahintaan 20:1, esimerkiksi ainakin 25:1.It has been found that in order to achieve better solubility and stability, the molar ratio of bile acid to peptide is at least 20: 1, for example at least 25: 1.
Taman keksinndn mukaiset yhdisteet on erityisen 30 toivottavaa vapauttaa kohteeseen pitkan ajanjakson aika-na, esimerkiksi viikosta vuoteen vaihtelevan ajanjakson aikana yhdesta ainoasta antamisesta. Voidaan kayttaa hy-vaksi erilaisia hitaasti vaikuttavaa ainetta vapauttavia depotistutteita tai ruiskeina annosteltavia muotoja. An-35 nostusmuoto voi esimerkiksi sisaitaa yhdisteen fysiologi-It is especially desirable to release the compounds of this invention over a long period of time, for example over a period of weeks to years, from a single administration. Various sustained release depot implants or injectable forms may be utilized. For example, the An-35 dosage form may contain a physiological
IIII
» · 21 Π r. *7 ιΛ S L·^ L Ο sesti hyvSksyttåvaa myrkytdnté suolaa, jonka liukoisuus ruumlin nesteisiin on plenl, eslmerklksl (a) moniemaksi-sen hapon, kuten fosforihapon, rikkihapon, sitruunahapon, viinihapon, parkkihapon, pamonihapon, algiinihapon, poly-5 glutamiinihapon, naftaleenimono- tai -disulfonihappojen, polygalakturonihapon tal vastaavan, muodostamaa happoad-ditiosuolaa; (b) moniarvoisten metallikationin, kuten slnkln, kalsiu-min, vismutin, bariumin, magneslumln, alumiinin, kuparln, 10 koboltin, nikkelin, kadmlumln tai vastaavan, tai esimer- kiksi N,N'-dibentsyylietyleenidiamiinista tai etyleeni-diamiinista muodostuneen orgaanisen kationin muodostamaa suolaa; tai (c) yhdistelm&å, joka koostuu (a):sta ja (b):stå, esimer- 15 kiksi sinkkitannaattisuolaa.»· 21 Π r. * 7 ιΛ SL · ^ Ο Ο a highly acceptable toxic salt having a solubility in body fluids of plenl, eslmerklksl (a) a polybasic acid such as phosphoric acid, sulfuric acid, citric acid, tartaric acid, tartaric acid, tannic acid, pamonic acid, alginic acid, alginic acid, alginic acid - an acid addition salt of polygalacturonic acid or -disulfonic acids, corresponding to polygalacturonic acid; (b) an organic cation of a polyvalent metal cation such as n, N'-dibenzylethylenediamine or ethylenediamine formed by a polyvalent metal cation such as slurry, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium or the like, or for example salt; or (c) a combination of (a) and (b), for example a zinc tannate salt.
LisSksi tåman keksinnOn mukaiset yhdisteet tai edullisesti, niiden suhteellisen liukenemattomat suolat, kuten esimerkiksi juuri edelld esitetyt suolat, voidaan formuloida ruiskeina annettavaksi soveltuvaksi geeliksi, 20 esimerkiksi alumiinimonostearaattigeeliksi seesamidJljyå kfiyttåen. Erityisen edullisia suoloja ovat sinkkisuolat, sinkkitannaattisuolat, pamoaattisuolat ja vastaavat.In addition, the compounds of this invention, or preferably, their relatively insoluble salts, such as the salts just set forth above, may be formulated into a suitable gel for injection, for example, an aluminum monostearate gel using sesame oil. Particularly preferred salts are zinc salts, zinc tannate salts, pamoate salts and the like.
Eråsta toista tyyppiS oleva hitaasti vaikuttavaa ainetta vapauttava depotformula, joka soveltuu annettavaksi ruis-... 25 keina tai istutettavaksi kudok seen, voisi sisåltaa yhdis-tetta tai sen suolaa dispergoituna tai kapseloituna hitaasti hajoavaan, myrkyttdmaan, ei-antigeeniseen polymee-riin, kuten esimerkiksi polymaitohappo-polyglykolihappo-polymeeriin. Yhdisteet tai, edullisesti, niiden suhteel-30 lisen liukenemattomat suolat, kuten esimerkiksi edelld esitetyt suolat, voidaan myds formuloida kolesterolimat-riksipelleteiksi tai silastomeerimatriksi-istutteiksi tai hydrogeeli- tai huokoskapseleiksi, erityisesti silloin, kun niita on tarkoitus kayttaa eiaimilia. Muut hitaasti 35 vaikuttavaa ainetta vapauttavat depotistute- ja ruiske- • · « 92326 22 formulat, esimerkiksi liposomit, ovat alan kirjallisuu-dessa hyvin tunnettuja. Viitataan esimerkiksi teokseen Sustained and Controlled Release Drug Delivery Systems, toim. J.R. Robinson, Marcel Dekker, Inc., New York, 5 1978. Erityisesti LHRH-tyyppisten yhdisteiden lahteeksi voidaan todeta esimerkiksi US-patenttijulkaisu 4 010 125. Tutustukaa myiis C.C. Pittin artikkeliin "The controlled delivery of polypeptides including LHRH analogs" teokses-sa LHRH and its Analogs: Contraceptive and Therapeutic 10 Applications, Part 2, toim. B.H. Vickery ja J.J. Nestor, Jr., MTP Press, Boston, 1987, s. 557.Another type S sustained release depot formulation suitable for administration as an injection or implant into a tissue could contain the compound or a salt thereof dispersed or encapsulated in a slowly degradable, toxic, non-antigenic polymer such as, for example. polylactic-polyglycolic acid polymer. The compounds or, preferably, their relatively insoluble salts, such as the salts set forth above, may be formulated as cholesterol matrix pellets or silastomer matrix implants or hydrogel or pore capsules, especially when not intended for use. Other sustained release depot and injection formulations, for example liposomes, are well known in the art. Reference is made, for example, to Sustained and Controlled Release Drug Delivery Systems, ed. J.R. Robinson, Marcel Dekker, Inc., New York, 5 1978. In particular, the source of LHRH-type compounds can be found in U.S. Patent 4,010,125. See also C.C. Pitt's article "The controlled delivery of polypeptides including LHRH analogs" in LHRH and its Analogs: Contraceptive and Therapeutic Applications, Part 2, ed. B. H. Vickery and J.J. Nestor, Jr., MTP Press, Boston, 1987, p. 557.
Peptidien syntetisointiSynthesis of peptides
TamSn keksinnOn mukaisia polypeptideja voidaan syntetisoida peptidi-alaan perehtyneille tutuin menette-15 lytavoin. Erinomainen yhteenveto monista sellaisista kayttiikelpoisista menettelytavoista on ldydettavissa teoksista J.M. Stewart ja J.D. Young, Solid Phase Peptide Synthesis, W.H. Freeman Co., San Francisco, 1969 ja J. Meienhofer, Hormonal Prosteins and Peptides, osa 2, Aca-20 demic Press, New York, 1973, s. 46, kun kysymyksessa on peptidien kiintea-faasisynteesi, ja teoksesta E. Schroder ja K. Lubke, The Peptides, osa 1, Academic Press, New York, 1865, kun kysymyksessa on klassinen liuossynteesi.The polypeptides of the present invention can be synthesized by methods familiar to those skilled in the art of peptide. An excellent summary of many such useful procedures can be found in J.M. Stewart and J.D. Young, Solid Phase Peptide Synthesis, W.H. Freeman Co., San Francisco, 1969 and J. Meienhofer, Hormonal Prosteins and Peptides, Volume 2, Aca-20 demic Press, New York, 1973, p. 46, in the case of solid-phase synthesis of peptides, and E. Schroder and K. Lubke, The Peptides, Part 1, Academic Press, New York, 1865, when it comes to classical solution synthesis.
Yleensa nama menetelmat kasittavat yhden tai .. 25 useamman aminohapon tai sopivalla tavalla substituoidun aminohapon liittamisen perakkain kasvavaan peptidiket-juun. Normaalisti joko ensimmaisen aminohapon aminoryhma tai karboksyyliryhma suojataan sopivalla suojausryhmaiia. Suojattu tai johdannaisekseen muunnettu aminohappo voi-30 daan sitten joko kiinnittaa inerttiin kiinteaan kantajaan tai kayttaa hyvaksi liuoksena liittamaiia jarjestyksessa seuraava aminohappo, jossa vapaa ryhma (amino- tai karboksyyliryhma) on sopivalla tavalla suojattu, amidisidok-sen muodostamiseen soveltuvissa olosuhteissa. Sen jaikeen 35 tasta vastaliitetysta aminohappotahteesta poistetaan suo- 92326 23 jausryhma, ja sitten liitetaan seuraava (sopivalla taval-la suojattu) aminohappo jne. Sen jalkeen kun kaikki ami-nohapot on liitetty oikeassa jarjestyksessa, kaikki jai-jelia olevat suojausryhmat (ja mahdollinen kiintea kan-5 taja) poistetaan peråkkåin tai samanaikaisesti, jolloin saadaan lopullinen polypeptidi. Muuntamalla tata yleista menettelyå yksinkertaisella tavalla on mahdollista lisåta kasvavaan ketjuun enemmSn kuin yksi aminohappo kerral-laan, esimerkiksi liittamaiia toisiinsa suojattu tripep-10 tidi ja sopivalla tavalla suojattu dipeptidi (sellaisissa olosuhteissa, etta asymmetriakeskukset eivat rasemisoi-du), jolloin muodostuu suojausten poiston jalkeen penta-peptidi.In general, these methods involve the sequential addition of one or more amino acids or an appropriately substituted amino acid to a growing peptide chain. Normally, either the amino group or the carboxyl group of the first amino acid is protected with a suitable protecting group. The protected or derivatized amino acid can then either be attached to an inert solid support or utilize as a solution the next amino acid in the sequence in which the free group (amino or carboxyl group) is suitably protected under conditions suitable for amide bond formation. From this newly linked amino acid residue of its verse 35, the protecting group 92326 23 is removed, and then the next (appropriately protected) amino acid is added, etc. After all amino acids are attached in the correct order, all protecting groups (and any solid moieties) are attached. -5) is removed sequentially or simultaneously to give the final polypeptide. By modifying this general procedure in a simple manner, it is possible to add more than one amino acid at a time to the growing chain, for example a tripept-10 protected peptide and a suitably protected dipeptide (under conditions such that the centers of asymmetry are not racemized) to form protection. peptide.
Synteesin edullisia toteutustapoja 15 Eras erityisen edullinen menetelma taman keksinnOn mukaisten yhdisteiden valmistamiseksi sisaitaa kiintea-faasipeptidisynteesin.Preferred Embodiments of the Synthesis A particularly preferred method for preparing the compounds of this invention involves solid phase peptide synthesis.
Tåssa erityisen edullisessa menetelmassa aminohappo j en funktionaalinen a-aminoryhma suojataan happo- tai 20 emasherkaiia ryhmålia. Sellaisten suojausryhmien tulisi olla ominaisuuksiltaan stabiileja peptidisidosten muodos-tusolosuhteissa ja samalla helposti poistettavissa hajot-tamatta kasvavaa peptidiketjua tai rasemisoimatta mitaan sen sisaitamista asymmetriakeskuksista. Sopivia suojaus-.. 25 ryhmia ovat t-butyloksikarbonyyli (Boc), bentsyloksikar-bonyyli (Cbz), bifenyyli-isopropyloksikarbonyyli, t-amy-loksikarbonyyli, isobornyloksikarbonyyli, a,a-dimetyyli- 3,5-dimetoksibentsyloksikarbonyyli, o-nitrofenyylisulfe-nyyli, 2-syaani-t-butyloksikarbonyyli, 9-fluorenyylimety-30 loksikarbonyyli ja vastaavat, erityisesti t-butyloksikar-bonyyli.In this particularly preferred method, the α-amino functional group of the amino acids is protected by acid or 20 female-sensitive groups. Such protecting groups should be stable in properties under peptide bond formation conditions while being easily removed without disrupting the growing peptide chain or racemizing any of the asymmetric centers it contains. Suitable protecting groups include t-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz), biphenylisopropyloxycarbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl, α, α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, o-nitrile nyl, 2-cyano-t-butyloxycarbonyl, 9-fluorenylmethoxycarbonyl and the like, especially t-butyloxycarbonyl.
Erityisen edullisia ryhmia sivuketjujen suojauk-seen ovat arginiinin tapauksessa nitro, p-tolueenisulfo-nyyli, 4-metoksibentseenisulfonyyli, Cbz, Boc ja adaman-35 tyloksikarbonyyli; tyrosiinin tapauksessa bentsyyli, o-bromibentsyloksikarbonyyli, 2,6-diklooribentsyyli, iso- 24 propyyli, sykloheksyyli, syklopentyyli ja asetyyli; seriinin tapauksessa bentsyyli ja tetrahydropyranyyli; seka histidiinin tapauksessa bentsyyli, p-tolueenisulfo-nyyli ja 2,4-dinitrofenyyli.Particularly preferred groups for side chain protection in the case of arginine are nitro, p-toluenesulfonyl, 4-methoxybenzenesulfonyl, Cbz, Boc and adaman-35 tyloxycarbonyl; in the case of tyrosine, benzyl, o-bromobenzyloxycarbonyl, 2,6-dichlorobenzyl, isopropyl, cyclohexyl, cyclopentyl and acetyl; in the case of serine, benzyl and tetrahydropyranyl; and in the case of histidine, benzyl, p-toluenesulfonyl and 2,4-dinitrophenyl.
5 C-terminaalinen aminohappo kiinnitetåån sopivaan kiinteaan kantajaan. Sopivia kiinteita kantajia, joita voidaan kåyttåå edellå esitetyssa synteesisså, ovat sel-laiset aineet, jotka ovat inerttejå vaiheittaisten kon-densaatiosuojauksenpoistoreaktioiden reagensseille ja 10 olosuhteille ja jotka eivåt liukene kaytettaviin våliai-neisiin. Sopivia kiinteita kantajia ovat kloorimetyyli-polystyreeni-divinyylibentseenipolymeeri, hydroksimetyy-lipolystyreeni-divinyylibentseenipolymeeri ja vastaavat, erityisesti kloorimetyylipolystyreenistå ja 1 %:ista divi-15 nyylibentseenia koostuva polymeeri. Siina erikoistapauk-sessa, ettå yhdisteen C-terminaalinen tahde on glysiin-amidi, yksi erityisen kayttokelpoinen kantaja on bents-hydryyliaminopolystyreeni-divinyylibentseenipolymeeri, jota kuvaavat P. Rivaille et al., Helv. Chim. Acta 54 20 (1971) 2772. Kiinnitys kloorimetyylipolystyreeni-divi- nyylibentseenityyppiseen hartsiin toteutetaan antamalla N^-suojatun amihohapon, erityisesti Boc-aminohapon rea-goida cesium-, trimetyyliammonium-, trietyyliammonium-tai 1,5-diatsabisyklo,^4.4.07undek-5-eenisuolanaan tai ·' 25 muuna samankaltaisena suolana etanolissa, asetonitriilis-så, Ν,Ν-dimetyyliformamidissa (DMF) tai vastaavassa, erityisesti cesiumsuolana DMF:ssa, kloorimetyylihartsin kanssa korotetussa lampQtilassa, esimerkiksi noin 40-60°C: ssa, edullisesti noin 50°C:ssa, noin 12-48 tuntia, edul-30 lisesti noin 24 tuntia. N°^-Boc-aminohappo kiinnitetaan ·· bentshydryyliamiinihartsiin N,N'-di-isopropyylikarbodi- imidin (DIC)/1-hydroksibentsotriatsolin (HBT) vålittamån, noin 2-24 tuntia, edullisesti noin 12 tuntia, kestavan kytkentåreaktion avulla jossakin liuottimessa, kuten di-35 kloorimetaanissa tai DMF:sså, edullisesti dikloorimetaa- • 25 32326 nissa, noin 10-50°C:n, edullisesti 25°C:n, lampotilas-sa. Peråkkaisten suojattujen aminohappojen liittaminen voidaan toteuttaa automaattisessa polypeptidien synte-tisointilaitteessa, joka on alalia tunnettu. N^-suojaus-5 ryhmien poisto voidaan toteuttaa esimerkiksi trifluori-etikkahapon metyleenikloridiliuoksella, vetykloridin di-oksaaniliuoksella, vetykloridin etikkahappoliuoksella tai jollakin muulla vahvan hapon liuoksella, edullisesti 50 %:isella trifluorietikkahapon dikloorimetaaniliuok-10 sella, suunnilleen ymparistdn lampotilassa. Kutakin suo-jattua aminohappoa lisataan edullisesti suunnilleen 2,5-kertaisesti ylimaarin ja liittaminen voidaan toteuttaa dikloorimetaanissa, dikloorimetaani-DMF-seoksessa tai vastaavassa,erityisesti dikloorimetaanissa, suunnilleen 15 ympariston lampotilassa. Kytkentaalne on normaalisti DCC dikloorimetaanissa mutta voi olla myos N, N*-di-isopropyy-likarbodi-imidi (DIC) tai muu karbodi-imidi joko yksi-nåån tai yhdessa HBT:n, N-hydroksisukkinimidin, muun N-hydroksi-imidin tai oksiimin kanssa. Vaihtoehtoisesti 20 voidaan kåyttaa suojattuja aminohappojen aktiivisia es-tereita (esim. nitrofenyyli- ja pentafluorifenyylieste-ria ja vastaavia) tai symmetrisia anhydrideja.The 5 C-terminal amino acid is attached to a suitable solid support. Suitable solid carriers which can be used in the above synthesis are those which are inert to the reagents and conditions of the stepwise condensation deprotection reactions and which are insoluble in the vehicles used. Suitable solid carriers include chloromethyl-polystyrene-divinylbenzene polymer, hydroxymethylpolystyrene-divinylbenzene polymer and the like, especially a polymer consisting of chloromethylpolystyrene and 1% divinylbenzene. In the special case that the C-terminal residue of the compound is glycinamide, one particularly useful carrier is the benzhydrylaminopolystyrene-divinylbenzene polymer described by P. Rivaille et al., Helv. Chim. Acta 54 20 (1971) 2772. Attachment to a chloromethylpolystyrene-divinylbenzene type resin is accomplished by reacting an N4-protected amino acid, especially a Boc amino acid, with cesium, trimethylammonium, triethylammonium or 1,5-diazabecyclo, As its 5-ene salt or other similar salt in ethanol, acetonitrile, Ν, Ν-dimethylformamide (DMF) or the like, especially as the cesium salt in DMF, at elevated lamp temperature with chloromethyl resin, for example at about 40-60 ° C, preferably about At 50 ° C, about 12-48 hours, preferably about 24 hours. The N, N'-diisopropylcarbodiimide (DIC) / 1-hydroxybenzotriazole (HBT) is coupled to the benzhydrylamine resin by a coupling reaction in a solvent for about 2-24 hours, preferably about 12 hours, such as di-35 in chloromethane or DMF, preferably dichloromethane, at a temperature of about 10-50 ° C, preferably 25 ° C. The incorporation of successive protected amino acids can be accomplished in an automated polypeptide synthesizer known in the art. The removal of the N-protecting groups can be carried out, for example, with a methylene chloride solution of trifluoroacetic acid, a dioxane solution of hydrogen chloride, an acetic acid solution of hydrochloric acid or another strong acid solution, preferably a 50% solution of trifluoroacetic acid in dichloromethane, at approximately ambient temperature. Each protected amino acid is preferably added approximately 2.5 times the excess and the coupling may be carried out in dichloromethane, dichloromethane-DMF or the like, especially dichloromethane, at approximately ambient temperature. The coupling is normally DCC in dichloromethane but may also be N, N * -diisopropylcarbodiimide (DIC) or another carbodiimide, either alone or in combination with HBT, N-hydroxysuccinimide, other N-hydroxyimide or with oxime. Alternatively, protected active esters of amino acids (e.g., nitrophenyl and pentafluorophenyl ester and the like) or symmetrical anhydrides may be used.
Kiinteafaasisynteesin lopussa taydellisesti suo-jattu polypeptidi irrotetaan hartsista. Polypeptidin ja • · • 25 hartsikantajan valisen sidoksen ollessa bentsyyliesteri- tyyppia lohkaisu toteutetaan aminolyysilla, joka suori-tetaan peptidien, jotka sisSltavat C-terminaalisena ami-nohappona proliinin, tapauksessa alkyyliamiinilla tai fluorialkyyliamiinilla, tai aminolyysilla, joka suori-30 tetaan peptidien, jotka sisaltSvat C-terminaalisena ami- * nohappona glysiinin, tapauksessa esimerkiksi ammoniakki-metanoli- tai ammoniakki-etanoliyhdistelmailS, noin 10-50°C:n, edullisesti noin 25°C:n, lampdtilassa reaktio-ajan ollessa noin 12-24 tuntia, edullisesti noin 18 tun- 35 tia. Vaihtoehtoisesti peptidi voidaan irrottaa hartsista ' · 92326 26 transesteroinnin avulla, jota suoritetaan esimerkiksi metanolilla ja jota seuraa aminolyysi. Suojattu peptidi voidaan tåsså vaiheessa puhdistaa silikageelikromatogra-fian avulla. Sivuketjuja suojaavien ryhmien poisto poly-5 peptidistå toteutetaan kåsittelemållå aminolyysituote esimerkiksi vedettomållå nestemåisellå vetyfluoridilla anisolin tai jonkin muun karboniumakseptorin ollessa mu-kana, vetyfluoridipyridiinikompleksilla tai tris(trifluo-riasetyyli)boorilla ja trifluorietikkahapolla, pelkistå-10 målla se vedyllå palladium-hiili- tai palladium-polyvi-nyylipyrrolidonikatalysaattorin ollessa mukana tai pel-kiståmållå se natriumilla nestemåisesså ammoniakissa, edullisesti nestemåisellå vetyfluoridilla ja anisolilla noin -10 - +10°C:n, edullisesti noin 0°C:n, låmpotilas-15 sa kåsittelyajan ollessa noin 15 minuutista 1 tuntiin, edullisesti noin 30 minuuttia. Kun kysymyksesså ovat glysiiniterminaaliset aminohapot bentshydryyliamiini-hartseilla, hartsin lohkaisu ja suojauksen poisto voidaan yhdiståå yhdeksi ainoaksi vaiheeksi, jossa kåyte-20 tåån hyvåksi nestemåistå vetyfluoridia ja anisolia kuten edellå on kuvattu. Polypeptidi, josta suojaukset on poistettu tåydellisesti, puhdistetaan sitten kromatografisten vaiheiden sarjalla, jossa kåytetåån jotakin tai kaikkia seuraavista vaihetyypeistå: ioninvaihtokromato-\ 25 grafia-asetaattimuodossa olevalla heikosti emåksisellå hartsilla; hydrofobinen adsorptiokromatografia derivati-soimattomalla polystyreenidivinyylibentseenihartsilla (esimerkiksi Amberlite XAD:llå); silikageeliadsorptio-kromatografia; ioninvaihtokromatografia karboksimetyyli-30 selluloosalla; partitiokromatografia, esimerkiksi Sep-hadex G-25 vastavirtadistribuutio; ja suuren erotusky-vyn nestekromatografia (HPLC), erityisesti kåanteis-faasi-HPLC kåyttåen kolonnin sidottu faasi - tåytteenå oktyyli- tai oktadekyylisilyylisilikaa.At the end of solid phase synthesis, the fully protected polypeptide is cleaved from the resin. When the bond between the polypeptide and the resin support is of the benzyl ester type, cleavage is accomplished by aminolysis in the case of peptides containing proline as the C-terminal amino acid with alkylamine or fluoroalkylamine, or by aminolysis of peptides containing proline. As the C-terminal amino acid in the case of glycine, for example ammonia-methanol or ammonia-ethanol combinations, at a lamp temperature of about 10-50 ° C, preferably about 25 ° C, with a reaction time of about 12-24 hours, preferably about 18 hours 35 hours. Alternatively, the peptide can be cleaved from the resin by transesterification, for example with methanol, followed by aminolysis. At this stage, the protected peptide can be purified by silica gel chromatography. Removal of side chain protecting groups from the poly-5 peptide is accomplished by treating the aminolysis product with, for example, anhydrous liquid hydrogen fluoride in the presence of anisole or another carbonium acceptor, palladium-palladium-hydrogen-trifluoroacetic acid or tris (trifluoroacetate) fluoroacetylacarboxylate, in the presence of a polyvinylpyrrolidone catalyst or by reducing it with sodium in liquid ammonia, preferably liquid hydrogen fluoride and anisole at a temperature of about -10 to + 10 ° C, preferably about 0 ° C, for about 15 minutes at a temperature of about 15 minutes. preferably about 30 minutes. In the case of glycine-terminal amino acids on benzhydrylamine resins, cleavage and deprotection of the resin can be combined into a single step in which the user-20 is favored with liquid hydrogen fluoride and anisole as described above. The deprotected polypeptide is then purified by a series of chromatographic steps using one or all of the following types of steps: with a weakly basic resin in the form of ion exchange chromatography; hydrophobic adsorption chromatography on non-derivatized polystyrene divinylbenzene resin (e.g., with Amberlite XAD); silikageeliadsorptio chromatography; ion exchange chromatography on carboxymethyl-30 cellulose; partition chromatography, for example Sep-hadex G-25 countercurrent distribution; and high performance liquid chromatography (HPLC), especially reverse phase HPLC using column bound phase - packed with octyl or octadecylsilyl silica.
35 Kåytettåesså 1-, 2-, 3- tai 6-asemassa raseemista aminohappoa diastereomeeriset nonapeptidi- tai dekapep- qo 7 9 c y i. sj i. o 27 tidilopputuotteet erotetaan ja haluttu peptidi, joka si-saitaa asianmukaisessa asemassa D-aminohapon, eristetaan ja puhdistetaan, edullisesti edellS mainitun kromatografisen prosessin aikana.When a racemic amino acid is used at the 1-, 2-, 3- or 6-position, the diastereomeric nonapeptide or decapepto 7 9 cy i. Sj i. O 27 end products are separated and the desired peptide containing the D-amino acid at the appropriate position is isolated. and purified, preferably during the aforementioned chromatographic process.
5 Peptideina, jotka sisaitavat C-terminaalisen atsa- glysiiniamidin, valmistus toteutetaan edullisesti klas-sista peptidien liuossyntetisointia ja tunnettuja pepti-divaiituotteita kayttaen. Tata kuvataan yksityiskohtai-semmin esimerkissM 3.The preparation of peptides containing the C-terminal azaglycinamide is preferably carried out using classical solution synthesis of peptides and known peptide products. This is described in more detail in Example 3.
10 Kaavan (I) mukaisia yhdisteita ja niiden fysiolo gisesti hyvaksyttavia suoloja valmistetaan yleisesti siten, ettå suojatusta polypeptidista poistetaan suojaryh-mat ja mahdollisesti kovalenttisesti sitoutunut kiintea kantaja, kaavan (I) mukaisen yhdisteen tai sen suolan ai- 15 kaansaamiseksi; tai kaksi halutun, kaavan (I) mukaisen yhdisteen fragmenttia liitetaan yhteen vaadittavaan jar-jestykseen; tai (a) kaavan (I) mukainen yhdiste muunnetaan fysiologisesti hyvaksyttavaksi suolakseen; 20 (b) kaavaa (I) mukaisen yhdisteen suola muunnetaan fysio logisesti hyvaksyttavaksi suolaksi tai (c) kaavan (I) mukaisen yhdisteen suola muunnetaan va-paaksi polypeptidiksi, jolla on kaava (I).Compounds of formula (I) and their physiologically acceptable salts are generally prepared by deprotecting a protected polypeptide and optionally a covalently bonded solid support to provide a compound of formula (I) or a salt thereof; or two fragments of the desired compound of formula (I) are joined to one of the required sequences; or (a) converting a compound of formula (I) into a physiologically acceptable salt; (B) converting a salt of a compound of formula (I) into a physiologically acceptable salt or (c) converting a salt of a compound of formula (I) into a free polypeptide of formula (I).
Seuraavat esimerkit on esitetty, jotta alan ammat-. 25 ti-ihmiset kykenevat saamaan paremman kasityksen keksin-nOsta ja soveltamaan sita kaytannOssa. Niita ei tulisi kasittaa keksinnOn piiria rajoittaviksi, vaan pelkastaan keksintoa valaiseviksi ja kuvaaviksi.The following examples are provided to enable those skilled in the art. 25 Ti people are able to get a better understanding of the invention and apply it in practice. They should not be construed as limiting the scope of the invention, but merely as illustrating and illustrating the invention.
Valmistus APreparation A
30 3-(2-naftyyli)-DL-alaniini 3-(2-naftyyli)-DL-alaniini valmistetaan US-patent-tijulkaisussa nro 4 341 767 esitetylia menettelytavalla.3- (2-Naphthyl) -DL-alanine 3- (2-naphthyl) -DL-alanine is prepared by the procedure described in U.S. Patent No. 4,341,767.
N-asetyyli-3-(2-naftyyli)-DL-alaniinin valmistus ja muuntaminen metyyli-N-asetyyli-3-(2-naftyyli)-DL-ala- 35 ninaatiksi seka D-isomeerin erottaminen toteutetaan US- 92326 28 patenttijulkaisussa nro 4 341 767 esitetyllå menettely-tavalla.The preparation and conversion of N-acetyl-3- (2-naphthyl) -DL-alanine to methyl N-acetyl-3- (2-naphthyl) -DL-alaninate and the separation of the D-isomer are carried out in U.S. Pat. No. 9,232,268. 4,341,767.
Valmistus BPreparation B
Bentsyyli-N^-bentsyloksikarbonyyli-NfN'-guanidinodi-5 isopropyyli-D-homoargininaattitolueenisulfonaattiBenzyl N, N-benzyloxycarbonyl N, N'-guanidinodi-5-isopropyl-D-homoargininate toluenesulfonate
Seokseen, joka sisalsi 5,24 g bentsyyli-N°°-bentsyl-karbonyyli-D-lysinaattitolueenisulfonaattia (B. Bezus ja L. Zervas, J. Am. Soc . 83 (1961) 719) ja 1,72 ml di-iso-propyylietyyliamiinia 60 mlrssa dioksaania, lisattiin 10 1,89 g N,N'-di-isopropyylikarbodi-imidia. Reaktioseosta sekoitettiin 100°C:ssa 6 tuntia, ja se jåahdytettiin huo-neen låmpotilaan ja haihdutettiin, jolloin saatiin kiin-tea aine. Kiintoaine suspendoitiin 20 ml:aan lamminta DMF:a, suspensio suodatettiin N,N'-di-isopropyyliurean 15 poistamiseksi ja suodos haihdutettiin, jolloin saatiin kiintea aine. Bentsyyli-Nc^'-bentsyloksikarbonyyli-N,N'-guanidinodi-isopropyyli-D-homoargininaattitolueenisulfo-naatti saatiin valkoisena kiinteåna aineena kiteyttåmal-1M se metanoli-etyyliasetaattiseoksesta; 20 /o£7d = “7,26° (c = 0,3, MeOH).To a mixture of 5.24 g of benzyl-N-benzo-carbonyl-D-lysinate toluenesulfonate (B. Bezus and L. Zervas, J. Am. Soc. 83 (1961) 719) and 1.72 ml of di-iso -propylethylamine in 60 ml of dioxane, 1.89 g of N, N'-diisopropylcarbodiimide were added. The reaction mixture was stirred at 100 ° C for 6 hours and cooled to room temperature and evaporated to give a solid. The solid was suspended in 20 ml of warm DMF, the suspension was filtered to remove N, N'-diisopropylurea and the filtrate was evaporated to give a solid. Benzyl N, N'-benzyloxycarbonyl-N, N'-guanidinodiisopropyl-D-homoargininate toluenesulfonate was obtained as a white solid by crystallization from a 1M mixture of methanol and ethyl acetate; 20 DEG C. = 7.26 ° (c = 0.3, MeOH).
Vastaavasti kSyttamSlla edellå kuvattua menettely-tapaa mutta vaihtamalla N,N'-di-isopropyylikarbodi-imidin tilalle N,N1-disykloheksyylikarbodi-imidi; N,N'-di-(n-heksyyli)-karbodi-imidi; *'25 N ,N' -dietyylikarbodi-imidi; N,N'-di-(n-propyyli)-karbodi-imidi; N-isopropyylikarbodi-imidi; N-propyylikarbodi-imidi; N-(n-butyyli)-karbodi-imidi? . 30 N,N'-di-(n-butyyli)-karbodi-imidi; : Ν,Ν'-di-isobutyylikarbodi-imidi? Ν,Ν'-di-(n-pentyyli)-karbodi-imidi; N,N1-di-isopentyylikarbodi-imidi; N,N1-difenyylikarbodi-imidi; 35 N,N'-ditolyylikarbodi-imidi tai 92326 29Accordingly, using the procedure described above but replacing N, N'-diisopropylcarbodiimide with N, N1-dicyclohexylcarbodiimide; N, N'-di (n-hexyl) carbodiimide; * '25 N, N'-diethylcarbodiimide; N, N'-di (n-propyl) carbodiimide; N-diisopropylcarbodiimide; N-propyl carbodiimide; N- (n-butyl) carbodiimide? . N, N'-di- (n-butyl) carbodiimide; : Ν, Ν'-diisobutylcarbodiimide? Ν, Ν'-di (n-pentyl) carbodiimide; N, N1-di-isopentyylikarbodi-imide; N, N1-diphenylcarbodiimide-imide; 35 N, N'-ditolylcarbodiimide or 92326 29
1-(3-dimetyyliaminopropyyli)-3-etyylikarbodi-imidihydro-kloridi tai vastaava saadaan bentsyyli-N °^-bentsyloksi-karbonyyli-Ν,Ν'-guanidinodisykloheksyyli-D-homoargini-naattia, = 8,07° (c = 0,9, MeOH) , bentsyyli-N1- (3-Dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride or the like gives benzyl N, N'-benzyloxycarbonyl-Ν, Ν'-guanidinodicyclohexyl-D-homoarginate, = 8.07 ° (c = 0 , 9, MeOH), benzyl-N
5 bentsyloksikarbonyyli-N,N'-guanidinodietyyli-D-homoargi- ninaattia, bentsyyli-N -bentsyloksikarbonyyli-N-N'-gua-nidinodi-(n-propyyli)-D-homoargininaattia, ~ 8,07° (c = 0,9, MeOH), bentsyyli-N*·-bentsyloksikarbonyyli-N-guanidino-(n-propyyli)-D-homoargininaattia, 10 bentsyyli-N*-bentsyloksikarbonyyli-N-guanidino-(n-butyy- li)-D-homoargininaattia, bentsyyli-N * -bentsyloksikarbonyyli-N,N'-guanidinodi-(n-butyyli)-D-homoargininaattia, bentsyyli-N* -bentsyloksikarbonyyli-N,N-guanidinodi-iso-15 butyyli-D-homoargininaattia, bentsyyli-N*-bentsyloksikarbonyyli-N,N'-guanidinodi-(n-pentyyli)-D-homoargininaattia, bentsyyli-N* -bentsyloksikarbonyyli-N,N'-guanidinodifenyy-li-D-homoargininaattia, 20 bentsyyli-N *-bentsyloksikarbonyyli-N,N' -guanidinodimetyy- li-D-homoargininaattia, bentsyyli-N*-bentsyloksikarbonyyli-N,N'-guanidinodi-(n-heksyyli)-D-homoargininaattia ja bentsyyli-N* -bentsyloksikarbonyyli-N,N1-guanidinodi-iso-: 25 propyyli-D-argininaattia, /jpQ-q = -10,5° (c = 0,5, MeOH), bentseenisulfonaattisuoloinaan. Vastaavasti korvaamalla D-lysinaatti bentsyyli-N *-bentsyloksikarbonyyli-D-orni-tinaatilla voidaan saada aikaan vastaavia arginiinianalo-geja tolueenisulfonaattisuoloinaan.5 benzyloxycarbonyl-N, N'-guanidinodietyl-D-homoargininate, benzyl-N-benzyloxycarbonyl-N-N'-guanidinodi- (n-propyl) -D-homoargininate, 8,08.07 ° (c = 0 , 9, MeOH), benzyl N * · -benzyloxycarbonyl-N-guanidino- (n-propyl) -D-homoargininate, benzyl-N * -benzyloxycarbonyl-N-guanidino- (n-butyl) -D- homoargininate, benzyl N * -benzyloxycarbonyl-N, N'-guanidinodi- (n-butyl) -D-homoargininate, benzyl-N * -benzyloxycarbonyl-N, N-guanidinodiiso-15-butyl-D-homoargininate, benzyl- N * -benzyloxycarbonyl-N, N'-guanidinodi- (n-pentyl) -D-homoargininate, benzyl-N * -benzyloxycarbonyl-N, N'-guanidinodiphenyl-D-homoargininate, benzyl-N * -benzyloxycarbonyl- N, N'-guanidinodimethyl-D-homoargininate, benzyl-N * -benzyloxycarbonyl-N, N'-guanidinodi- (n-hexyl) -D-homoargininate and benzyl-N * -benzyloxycarbonyl-N, N1-guanidinodi- iso-: 25 propyl D-argininate, [α] 20 D = -10.5 ° (c = 0.5, MeOH), benzene sulfonate. Similarly, by substituting benzyl N * benzyloxycarbonyl D-orninate for D-lysinate, the corresponding arginine analogs can be obtained as their toluenesulfonate salts.
30 Valmistus C30 Preparation C
^^ Q|^^ Q |
Bentsyyli-N -bentsyloksikarbonyyli-N ,N -eteno-D-homoargininaattiBenzyl N-benzyloxycarbonyl N, N-etheno-D-homoargininate
Seokseen, joka sisålsi 15 ml tolueenia ja 15 ml t-BuOH:ta, lisattiin 2,71 g bentsyyli-N*-bentsyloksikar-35 bonyylilysinaattia ja 1,46 g 2-metyylitio-imidatsoliini- 30 92326 hydrojodidia (saatavissa Aldrichilta). Seoksen pH saadet-tiin suunnilleen arvoon 8 di-isopropyylietyyliamiinia lisååmållå, ja liuosta refluksoitiin 24 tuntia.To a mixture of 15 mL of toluene and 15 mL of t-BuOH were added 2.71 g of benzyl N * -benzyloxycarbon-35 carbonyl lysinate and 1.46 g of 2-methylthioimidazoline-92,9326 hydroiodide (available from Aldrich). The pH of the mixture was adjusted to approximately 8 by the addition of diisopropylethylamine, and the solution was refluxed for 24 hours.
Liuos vakevoitiin alipaineessa, ja jaannos pakat-5 tiin silikageelipylvaaseen (250 mg). Pylvas eluoitiin kåyttåen C^C^-MeOH-seosgradienttia suhteesta 19:1 suh-teeseen 7:3. Tuotetta sisaltavat jakeet tunnistettiin TLC:n avulla, yhdistettiin ja haihdutettiin kuiviin, jol-loin saatiin 2,9 g valkoista vaahtoa.The solution was stabilized under reduced pressure, and the portion was packed into a silica gel column (250 mg). The column was eluted using a C 1 -C 4 -MeOH mixture gradient from 19: 1 to 7: 3. The product-containing fractions were identified by TLC, combined and evaporated to dryness to give 2.9 g of a white foam.
10 2 g;:n era edellå mainittua tuotetta liuotettiin 50 ml:aan EtOH:ta, joka sisålsi 0,8 g katalysaattoria, joka sisålsi 10 % Pd:a hiilikantoaineella. Liuosta sekoi-tettiin Η2»·η alla 8 tuntia. Seos suodatettiin seliitin (piimaan) låpi ja suodos haihdutettiin kuiviin, jolloin G G1 15 saatiin 1,2 g N ,N -etanohomoarginiinia valkoisena vaah- tona.A 2 g portion of the above product was dissolved in 50 mL of EtOH containing 0.8 g of a catalyst containing 10% Pd on carbon. The solution was stirred at Η2 »· η for less than 8 hours. The mixture was filtered through celite (diatomaceous earth) and the filtrate evaporated to dryness to give 1.2 g of N, N-ethanohomoarginine as a white foam.
Samalla tavoin mutta kåyttåmållå guanylointiaineena S-metyyli-3,4,5,6-tetrahydro-2-pyrimidiinitiolia (Aldrich) G G1 saatiin N ,N -propanohomoarginiinia valkoisena vaahtona.In a similar manner but using S-methyl-3,4,5,6-tetrahydro-2-pyrimidinethiol (Aldrich) G G1 as the guanylating agent, N, N-propanohomoarginine was obtained as a white foam.
20 Vastaavasti mutta kåyttåmållå guanylointiaineena S-metyylibis-(2,2,2-trifluorietyyli)-tiouroniumjodidia G G1 saatiin N ,N -bis-(trifluorietyyli)-homoarginiinia (Bth) valkoisena vaahtona.Similarly, but using S-methylbis- (2,2,2-trifluoroethyl) -thouronium iodide G G1 as the guanylating agent, N, N-bis- (trifluoroethyl) -homoarginine (Bth) was obtained as a white foam.
Valmistus DPreparation D
25 Ν'** -t-butyloksikarbonyyli-N ,N ’ -guanidinodi-iso- propyyli-D-homoarginiinitolueenisulfonaatti Tåmå valmistusesimerkki valaisee N,N'-guanidinodi-substituoitujen D-homoarginiinien valmistamista tolueeni-sulfonaattiesiasteistaan.Ν '** t-Butyloxycarbonyl-N, N'-guanidinodiisopropyl D-homoarginine toluenesulfonate This preparation example illustrates the preparation of N, N'-guanidinodi-substituted D-homoarginines from their toluene sulfonate precursors.
30 Seosta, joka sisålsi bentsyyli-N^-bentsyloksikar- : bonyyli-N,N'-guanidinodi-isopropyyli-D-homoargininaatti- tolueenisulfonaattia (3,25 g) ja 100 mg katalysaattoria, joka sisålsi 10 % Pd:a hiilikantoaineella, 50 ml:ssa jååetikkaa, kåsiteltiin vetykaasulla ilmakehån paineessa 35 4 tuntia. Katalysaattori erotettiin suodattamalla seosA mixture of benzyl N, N'-benzyloxycarbonyl-carbonyl N, N'-guanidinodiisopropyl-D-homoargininate toluenesulfonate (3.25 g) and 100 mg of a catalyst containing 10% Pd on carbon was added. per ml of glacial acetic acid, was treated with hydrogen gas at atmospheric pressure for 35 4 hours. The catalyst was filtered off
IIII
3131
o t',"7 So t ', "7 S
o l 6 seliitin lapi, ja suodos haihdutettiin, jolloin saatiin kiinteåtå N,N'-guanidinodi-isopropyyli-D-homoarginiinito-lueenisulfonaattia. Liuokseen, joka sisalsi tatå yhdistet-ta (2,13 g) 60 ml:ssa dioksaani-vesiseosta (1:1), lisat-5 tiin 10 ml IN natriumhydroksidia ja 0,4 g magnesiumoksidia. Sen jalkeen tåhån seokseen lisattiin 1,1 g di-(t-butyv-li)-dikarbonaattia, ja sitå sekoitettiin huoneen lampo-tilassa 2 tuntia. Magnesiumsuola erotettiin suodattamalla ja suodos våkevditiin alipaineessa. Emaksinen liuos pes-10 tiin etanolilla, ja sen jalkeen sen pH saadettiin arvoon 2,5 natriumsulfaatilla. Hapan vesiliuos uutettiin etyyli-asetaatilla, ja etyyliasetaattiliuos kuivattiin magnesium-sulfaatilla. Kuivausaine poistettiin suodattamalla, ja suodos haihdutettiin. Kiteytys etyyliasetaatti-heksaani-15 seoksesta tuotti tulokseksi -t-butyloksikarbonyyli-N,N1 -guanidinodi-isopropyyli-D-homoarginiinitolueenisulfonaat-tia.The celite was evaporated to give a solid N, N'-guanidinodiisopropyl-D-homoarginine toluenesulfonate. To a solution of the compound (2.13 g) in 60 ml of a dioxane-water mixture (1: 1) was added 10 ml of 1N sodium hydroxide and 0.4 g of magnesium oxide. Then, 1.1 g of di- (t-butyl) dicarbonate was added to the mixture, and it was stirred at room temperature for 2 hours. The magnesium salt was filtered off and the filtrate was concentrated under reduced pressure. The basic solution was washed with ethanol and then adjusted to pH 2.5 with sodium sulfate. The acidic aqueous solution was extracted with ethyl acetate, and the ethyl acetate solution was dried over magnesium sulfate. The desiccant was removed by filtration and the filtrate was evaporated. Crystallization from ethyl acetate-hexane-15 gave t-butyloxycarbonyl-N, N1-guanidino diisopropyl-D-homoarginine toluenesulfonate.
Menettelemalla samalla tavalla mutta kayttamalla asianmukaisia tolueenisulfonaattiesiasteita voidaan val-20 mistaa muita Ν°ί-t-butyloksikarbonyyli-N,N'-guanidinodi-substituoituja D-homoarginiini- tai D-arginiiniyhdistei-tå.By proceeding in the same manner but using appropriate toluenesulfonate precursors, other Ν ° ί-t-butyloxycarbonyl-N, N'-guanidino di-substituted D-homoarginine or D-arginine compounds can be prepared.
Valmistus EPreparation E
-t-butyloksikarbonyyli-3-/4'-(1'-propyylipipe-• 25 ridyyli)y-D-alaniini 400 ml:aan absoluuttista etanolia lisattiin 4,6 g natriummetallia ja seosta kuumennettiin. Tulokseksi saa-tuun natriumetoksidiliuokseen lisattiin 21,.7 g dietyyli-(asetamidomalonaattia) ja 16,4 g 4-pikolyylikloridihydro-. 30 kloridia (Aldrich Chemical Co). Reaktioseosta kuumennettiin 100°C:ssa 4 tuntia, ja se jåShdytettiin, suodatet-tiin ja vakevoitiin alipaineessa. Seos siirrettiin sili-kageelipylvaaseen metyleenikloridi-metanoliseoksessa (19:1) , ja eluointi suoritettiin samalla seoksella. Tuo-35 te paikallistettiin nopeasti liikkuvaksi UV-positiivisek- « 32 92326 si tåplåksi silikageeliohutkerroskromatogrammissa (eluent-tina metyleenikloridi-metanoliseos suhteessa 19:1). Yh-distetyt jakeet vakevoitiin, jolloin saatiin tuote.t-butyloxycarbonyl-3- [4 '- (1'-propylpiperidyl) -γ-D-alanine To 400 ml of absolute ethanol was added 4.6 g of sodium metal and the mixture was heated. To the resulting sodium ethoxide solution were added 21.7 g of diethyl (acetamidomalonate) and 16.4 g of 4-picolyl chloride hydrochloride. 30 chloride (Aldrich Chemical Co). The reaction mixture was heated at 100 ° C for 4 hours and cooled, filtered and stabilized under reduced pressure. The mixture was applied to a silica gel column in methylene chloride-methanol (19: 1) and eluted with the same mixture. The product was localized to a fast moving UV-positive spot on a silica gel thin layer chromatogram (methylene chloride-methanol 19: 1 as eluent). The combined fractions were concentrated to give the product.
Edellisen kappaleen mukainen tuote liuotettiin 200 5 ml:aan etanolia, ja tata liuosta kåsiteltiin liuoksella, joka sisalsi 2,72 g natriumhydroksidia 40 ml:ssa vettå, 50°C:ssa 6 tuntia. Liuos tehtiin happamaksi 12 ml :11a 6 N HCl:a, se haihdutettiin kuiviin, ja jåannos sekoitet-tiin 200 ml:aan dioksaania. Suspensio suodatettiin, ja 10 suodosta refluksoitiin 2 tuntia. Liuos jaahdytettiin ja haihdutettiin kuiviin, jolloin saatiin etyyli-N^Casetyyli-3-(4-pyridyyli)-DL-alaniinia valkoisena kiinteanS aineena.The product of the previous paragraph was dissolved in 200 5 ml of ethanol, and this solution was treated with a solution of 2.72 g of sodium hydroxide in 40 ml of water at 50 ° C for 6 hours. The solution was acidified with 12 mL of 6 N HCl, evaporated to dryness, and the residue was stirred in 200 mL of dioxane. The suspension was filtered and 10 filtrates were refluxed for 2 hours. The solution was cooled and evaporated to dryness to give ethyl N, N-Cetyl-3- (4-pyridyl) -DL-alanine as a white solid.
Osa tastå N-asetyyliesterista liuotettiin uudel-leen kasittelemalla sita 200 ml :11a subtilisiini Carls-15 bergia (Sigma Chem. Co., Proteaasi VIII) seoksessa joka sisalsi 300 ml dimetyylisulfoksidia ja 400 ml 0,01 M KCl, (pH 7,2). 6 tunnin kuluttua uudelleen liukenemi-nen oli taydellista. Liuos laimennettiin 400 ml:11a vetta ja uutettiin neljalla 750 ml:n erallS etyyliasetaat-20 tia. Orgaaniset kerrokset yhdistettiin ja kuivattiin mag- nesiumsulfaatilla seka vakevoitiin, jolloin saatiin etyy-li-N^-asetyyli-S-(4-pyridyyli)-3-alaninaattia oljyna.A portion of this N-acetyl ester was redissolved by treating it with 200 ml of subtilisin Carls-15 berg (Sigma Chem. Co., Protease VIII) in a mixture containing 300 ml of dimethyl sulfoxide and 400 ml of 0.01 M KCl, (pH 7.2 ). After 6 hours, redissolution was complete. The solution was diluted with 400 ml of water and extracted with four 750 ml portions of ethyl acetate. The organic layers were combined and dried over magnesium sulfate and concentrated to give ethyl N, N-acetyl-S- (4-pyridyl) -3-alaninate as an oil.
Oljyn annettiin 1,22 g:n kanssa n-propyylibromi-dia 50 ml:ssa etanolia, minka jålkeen liuos haihdutettiin 25 kuiviin, jolloin saatiin etyyli-N0^-asetyyli-3-(1-propyy- lipyridinium-4-yyli)-D-alaninaattibromidia valkoisena hygroskooppisena kiintoaineena.The oil was treated with 1.22 g of n-propyl bromide in 50 ml of ethanol, after which the solution was evaporated to dryness to give ethyl-N, N-acetyl-3- (1-propylpyridinium-4-yl) - D-alaninate bromide as a white hygroscopic solid.
Tama valkoinen kiintea aine liuotettiin 200 ml:aan etanolia ja pelkistettiin vetyatmosfaårissa kayttaen ka-30 talysaattorina 100 mg yhdistelmaa, joka sisalsi 10 % Pd:a hiilikantoaineellao 18 tunnin pelkistyksen jalkeen kata-lysaattori erotettiin suodattamalla ja liuos haihdutettiin, jolloin saatiin etyyli-N"^ -asetyyli-3-/4'-(1'-propyyli-piperidyyli)^7~D-alaninaattia kullanruskeana kiinteana 35 aineena. Vapaa happo valmistettiin refluksoimalla etyyli-This white solid was dissolved in 200 ml of ethanol and reduced under a hydrogen atmosphere using 100 mg of a combination of 10% Pd on carbon as a catalyst. After 18 hours of reduction, the catalyst was filtered off and the solution was evaporated to give ethyl -acetyl 3- [4 '- (1'-propyl-piperidyl) -β-D-alaninate as a tan solid 35. The free acid was prepared by refluxing ethyl acetate.
IIII
92326 esteria 100 ml:ssa 6 H HCl:å 4 tuntia, jolloin saatiin 3- /4 1 - (1' -piperidyyli)_7-D-alaniinia valkoisena kiinteånå aineena.92326 ester in 100 mL of 6 H HCl for 4 hours to give 3- [4 '- (1'-piperidyl) -7-D-alanine as a white solid.
Vapaa happo liuotettiin 100 ml:aan dioksaani-vesi-5 seosta (1:1), ja liuokseen lisattiin 2 g di-(t-butyyli)-dikarbonaattia. pH pidettiin 9:nå pH-staatilla 1 N NaOH: ta lisååmållå. Kahden tunnin kuluttua reaktioseos vake-vditiin alipaineessa ja pestiin 100 ml :11a etyylieette-riå, ja vesikerros siirrettiin Amberlite XAD-2 hartsi-10 pylvaaseen (hydrofobinen hartsi). Pylvas eluoitiin 250 ml :11a vettå ja sen jalkeen 250 ml:lla etanoli-vesiseosta (1:1). Etanolieluaatit yhdistettiin ja haihdutettiin kui-viin, jolloin saatiin N -t-butyloksikarbonyyli-3-/41 -(1'-propyylipiperidyyli)7-D-alaniinia valkoisena kiinteå-15 na aineena.The free acid was dissolved in 100 ml of a dioxane-water-5 mixture (1: 1), and 2 g of di- (t-butyl) dicarbonate was added to the solution. The pH was maintained at 9 with a pH statate by the addition of 1 N NaOH. After 2 hours, the reaction mixture was concentrated under reduced pressure and washed with 100 mL of ethyl ether, and the aqueous layer was applied to an Amberlite XAD-2 resin-10 column (hydrophobic resin). The column was eluted with 250 ml of water and then with 250 ml of ethanol-water (1: 1). The ethanol eluates were combined and evaporated to dryness to give N-t-butyloxycarbonyl-3- [41- (1'-propylpiperidyl) 7-D-alanine as a white solid.
Menettelemållå samalla tavalla mutta korvaamalla 4- pikolyylikloridihydrokloridi 3-pikolyylikloridihydro-kloridilla saadaan valmistetuksi -t-butyloksikarbo-nyyli-3-^3' - (1' -propyylipiperididyyli)_7-D-alaniinia.By proceeding in the same manner but substituting 3-picolyl chloride hydrochloride for 4-picolyl chloride hydrochloride, t-butyloxycarbonyl-3- [3 '- (1'-propylpiperididyl) -7-D-alanine is prepared.
20 Valmistus F20 Preparation F
Boc-Gly-O-hartsi 4,9 g Boc-glysiinia liuotettiin seokseen, joka sisålsi 50 ml etanolia ja 50 ml tislattua vetta. Liuok-sen pH såadettiin arvoon 7 cesiumvetykarbonaatin vesi-.·. 25 liuoksella. Liuotin haihdutettiin sitten alipaineessa.Boc-Gly-O resin 4.9 g of Boc-glycine was dissolved in a mixture of 50 ml of ethanol and 50 ml of distilled water. The pH of the solution was adjusted to 7 with aqueous cesium bicarbonate. 25 solution. The solvent was then evaporated under reduced pressure.
Sen jalkeen kun jåånnostå oli kuivattu 18 tuntia suuressa alipaineessa, se liuotettiin 150 ml:aan kuivaa DMF:å. Liuokseen lisattiin 25 g kloorimetyloitua poly-styreenihartsia, joka sisålsi 1 % divinyylibentseeniå 30 (Merrifield* 25 mmol:a kloridia vastaava). Seosta ravis-!: tettiin 50°C:ssa 24 tuntia, se suodatettiin, ja hartsi pestiin sen jålkeen peråkkåin DMFillå, vedellå ja etano-lilla. Hartsia kuivattiin alipaineessa 3 vuorokautta, jolloin saatiin 28,34 g Boc-Gly-O-hartsia.After drying under reduced pressure for 18 hours, the residue was dissolved in 150 ml of dry DMF. To the solution was added 25 g of chloromethylated poly-styrene resin containing 1% divinylbenzene (equivalent to 25 mmol of Merrifield * chloride). The mixture was shaken at 50 ° C for 24 hours, filtered, and the resin was then washed successively with DMF, water and ethanol. The resin was dried under reduced pressure for 3 days to give 28.34 g of Boc-Gly-O resin.
34 9 2 3 2 634 9 2 3 2 6
Valmistus GPreparation G
A. S-metyyli-3,4,5,6-tetrahydro-2-pyrimidiini- tiolihydrojodidiA. S-Methyl-3,4,5,6-tetrahydro-2-pyrimidine thiol hydroiodide
Liuokseen, joka sisalsi 23,24 g 3,4,5, 6-tetrahydro-5 2-pyriraidiinitiolia 175 ml:ssa MeOH:ta, lisattiin 15,57 ml Mel:ta, ja seosta refluksoitiin 1,5 tuntia. Liuotin haihdutettiin alipaineessa ja jaannos suspendoitiin di-etyylieetteriin. Sakka erotettiin suodattamalla ja kui-vattiin alipaineessa, jolloin saatiin 51,4 g S-metyyli-10 3,4,5,6-tetrahydro-2-pyrimidiinitiolia valkoisina kiteina.To a solution of 23.24 g of 3,4,5,6-tetrahydro-5 2-pyriraidinthiol in 175 mL of MeOH was added 15.57 mL of Mel, and the mixture was refluxed for 1.5 hours. The solvent was evaporated under reduced pressure and the portion was suspended in diethyl ether. The precipitate was filtered off and dried under reduced pressure to give 51.4 g of S-methyl-3,4,4,6-tetrahydro-2-pyrimidinethiol as white crystals.
B. N^-t-butyloksikarbonyyli-N ,N -propeno-L-ho- moarginiini S-metyyli-3,4,5,6-tetrahydro-2-pyrimidiinitioli saatiin 51,4 g:sta HI-suolaansa suorittamalla partitio 15 CH2Cl2:n (300 ml) ja 4 N NaOH:n (50 ml) kesken. Tulok- seksi saatu CH2Cl2-liuos vakevoitiin noin 100 ml:n tila-vuuteen, ja siihen lisattiin noin 100 ml EtOHrta. S-me-tyyli-3,4,5,6-tetrahydro-2-pyrimidiinitioliliuos lisSt-tiin pisaroittain 60°C:ssa liuokseen, joka sisalsi 24 g 20 lysiinihydrokloridia 66 ml:ssa 2 N NaOH:ta. Sekoittamista jatkettiin yon yli 60°C:ssa typen alla. 10,78 g lisaa HI-suolaa hajotettiin 15 ml:11a 4 N NaOH:ta, uutettiin 90 %:sella CH2Cl2:lla ja lisattiin pisaroittain ja se-koittaen toiset 24 tuntia 60°C:ssa, jona aikana reaktio : 25 meni suurin piirtein loppuun.B. N, N-t-Butyloxycarbonyl-N, N-propeno-L-homoarginine S-methyl-3,4,5,6-tetrahydro-2-pyrimidinethiol was obtained from its 51.4 g of its HI salt by partitioning. Between CH 2 Cl 2 (300 mL) and 4 N NaOH (50 mL). The resulting CH 2 Cl 2 solution was stabilized to a volume of about 100 mL, and about 100 mL of EtOH was added. A solution of S-methyl-3,4,5,6-tetrahydro-2-pyrimidinothiol was added dropwise at 60 ° C to a solution of 24 g of lysine hydrochloride in 66 ml of 2 N NaOH. Stirring was continued overnight at 60 ° C under nitrogen. 10.78 g of additional HI salt was decomposed with 15 mL of 4 N NaOH, extracted with 90% CH 2 Cl 2, and added dropwise with stirring for another 24 h at 60 ° C, during which time the reaction: 25 went to maximum roughly complete.
Reaktioseos pestiin kahdella EtOAc-eralla, ja Pha:ta sisaltava vesikerros laimennettiin 200 ml:11a dioksaania ja 200 ml:11a vetta. Liuos jaahdytettiin 0°C: seen, ennen kuin siihen lisattiin 33 mg di(t-butyyli)-30 dikarbonaattia ja 6 g MgO:ta. 4 g:n lisaera MgO:ta ja 22 g:n lisaera di-(t-butyyli)-dikarbonaattia saivat reaktion menemaan loppuun.The reaction mixture was washed with two portions of EtOAc, and the aqueous layer containing Pha was diluted with 200 mL of dioxane and 200 mL of water. The solution was cooled to 0 ° C before 33 mg of di (t-butyl) -30 dicarbonate and 6 g of MgO were added. An additional 4 g of MgO and an additional 22 g of di- (t-butyl) dicarbonate made the reaction complete.
MgO erotettiin suodattamalla seliitin lapi, ja suodos vakevoitiin alipaineessa puoleen tilavuudestaan.The MgO was separated by filtration through celite, and the filtrate was stabilized under reduced pressure to half its volume.
35 JaannSs pestiin kahdesti dietyylieetterilla ennen siir-35 JaannSs was washed twice with diethyl ether before
IIII
35 9 2 3 2 6 tåmistå silikageelipylvååseen (750 g silikageeliå, joka oli pakattu pylvaaseen CH^CNissa). Pylvas pestiin 5 lit-ralla CH^CN:. ja eluoitiin CH^CNrlla, joka sisalsi 10 % vetta (yht. 2 litraa), ja edelleen CH^CNtlla, joka sisal-5 si 20 % vetta (yht. 5 litraa). Tuotejakeet paikallistet-tiin ohutkerroskromatografisesti silikageelilevyilla (CH3CN-H0Ac-H20-seos suhteessa 4:1:1). Tuotejakeet yhdis-tettiin ja haihdutettiin, jolloin saatiin 5,0 g tuotetta valkoisena vaahtona, jonka sulamispiste oli 96°C ja 10 = 16,1° (c = 0,54, MeOH), ja lisaksi 15 g lievasti epa- puhdasta oljya.35 9 2 3 2 6 onto a silica gel column (750 g of silica gel packed in a column in CH 2 Cl 2). The column was washed with 5 liters of CH 2 Cl 2. and eluted with CH 2 Cl 2 containing 10% water (total 2 liters), and further with CH 2 CN containing 20% water (total 5 liters). The product fractions were localized by thin layer chromatography on silica gel plates (CH 3 CN-HOAc-H 2 O 4: 1: 1). The product fractions were combined and evaporated to give 5.0 g of product as a white foam, m.p. 96 ° C and 10 = 16.1 ° (c = 0.54, MeOH), and an additional 15 g of a slightly crude oil.
Samalla tavalla mutta kåyttamalla asianmukaisia S-metyyli-N,N1-dialkyylitiouroniumhydrojodideja saadaan vastaavia -t-butyloksikarbonyyli-N ,N -dialkyylihomo-15 arginiineja.In a similar manner but using the appropriate S-methyl-N, N1-dialkylthiouronium hydroiodides, the corresponding t-butyloxycarbonyl-N, N-dialkylhomo-15 arginines are obtained.
Esiinerkki 1 N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-hArg(CH2CF3)2~ D-Tyr-Leu-hArg(CH2CF3)2~Pro-D-Ala-NH2 Otsikon mukainen yhdiste on esitetty tassa seli-20 tyksessa myQs N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Bth- D-Tyr-Leu-Bth-Pro-D-Ala-NH2:na.Example 1 N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-hArg (CH2CF3) 2 ~ D-Tyr-Leu-hArg (CH2CF3) 2 ~ Pro-D- Ala-NH2 The title compound is shown in this specification also as N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Bth-D-Tyr-Leu-Bth- Pro-D-Ala-NH 2, respectively.
Beckman 990 Peptide Synthesizer-laitteen reaktio-astiaan laitettiin 0,758 g (0,5 mmol) bentshydryyliamino-polystyreenihartsia (Peninsula Labs., 0,66 mmol/g).0.758 g (0.5 mmol) of benzhydrylamino-polystyrene resin (Peninsula Labs., 0.66 mmol / g) was charged to the reaction vessel of the Beckman 990 Peptide Synthesizer.
25 Ensimmainen aminohappo liitettiin lisaamalla 0,284 gThe first amino acid was added by adding 0.284 g
Boc-D-Ala-OH:ta, 0,202 g Hbt:t. ja 3 ml 0,5 M di-iso-propyylikarbodi-imidiå. 3 tunnin liitSntajakson ja hart-sin pesun jalkeen tShSn hartsiin lisåttiin perakkain muut aminohapot synteesiohjelman mukaisesti seuraavasti: 30 Vaihe 1 CH2CL2~pesu kerran 1,5 min 2 suojauksen poisto CF3C02H-CH2Cl2-seoksella (1:1) kerran 1,5 min 3 suojauksen poisto cf3co2h-ch2ci2- 35 seoksella (1,1) kerran 30 min 36 92326 4 C^C^-pesu kolmesti 1,5 min 5 trietyyliamiini-Ci^C^-seos (1:1) kahdesti 1,5 min 6 CH2Cl2-pesu kolmesti 1,5 min 7 N -Roc-aminohappo liuotettuna 5 C^C^-DMF-seokseen (1:1) kerran lisays 8 N,N'-di-isopropyylikarbodi- imidiliuos (0,5 M) kerran lisays 9 Cl^C^-huuhtelu ja -såilytys, liitanta kerran liitanta- 10 reaktio 2 tuntia 10 CH2Cl2-huuhtelu kerran 1,5 min 11 CH2Cl2-pesu kolmesti 1,5 min 12 etanolipesu kolmesti 1,5 min 15 13 CHjC^-pesu kolmesti 1,5 minBoc-D-Ala-OH, 0.202 g Hbt. and 3 ml of 0.5 M diisopropylcarbodiimide. After a 3 hour addition period and washing of the resin, other amino acids were added sequentially to the tShS resin according to the synthesis program as follows: Step 1 CH 2 Cl 2 ~ wash once 1.5 min 2 deprotection with CF 3 CO 2 H-CH 2 Cl 2 (1: 1) once 1.5 min 3 protection removal with cf3co2h-ch2ci2-35 (1.1) once 30 min 36 92326 4 Cl2Cl2 wash three times 1.5 min 5 triethylamine-Cl2C2 mixture (1: 1) twice 1.5 min 6 CH2Cl2 washing three times for 1.5 min 7 N -Roc-amino acid dissolved in 5 ° C-CH 2 -DFF mixture (1: 1) once with 8 N, N'-diisopropylcarbodiimide solution (0.5 M) once with 9 Cl 2 Cl 2 rinse and storage, addition once addition reaction 2 hours 10 CH 2 Cl 2 rinse once 1.5 min 11 CH 2 Cl 2 wash three times 1.5 min 12 ethanol wash three times 1.5 min 15 13 CH 2 Cl 2 wash three times 1.5 min
Vaiheet 1-13 paattavat yhden aminohapon liitanta-syklin, ja reaktion loppuunmeno voidaan tarkistaa nin-hydriinimenetelmalla /E. Kaiser et al., Anal. Biochem.Steps 1-13 terminate the single amino acid addition cycle, and the completion of the reaction can be checked by the nin-hydrine method / E. Kaiser et al., Anal. Biochem.
34 (1970) 595J.34 (1970) 595J.
20 Hartsiin liitettiin aminohappoja perakkain kayt- taen 2,0-3,0 -kertaista moolista ylimaaraa kutakin suo-jattua aminohappoa sekå DIC:ta. Hartsi kSsiteltiin pe-rakkaisten liitantåsyklien aikana 0,269 g:11a Boc-Pro-OH:ta, 25 0,610 g:11a Boc-Bth-OH:ta, 0,311 g:11a Boc-Leu-OH’I^C^ta, 0,375 g:11a Boc-D-Tyr-OH:ta, 0,610 g:11a Boc-Bth-OH:ta, 0,380 g:11a Boc-Ser(bentsyyli)-OH:ta, ; 30 0,320 g:11a Boc-D-Pal(3)-OH:ta, 0,390 g:11a Boc-D-pCl-Phe-OH:ta, 0,400 g:11a Boc-D-Nal(2)-OH:ta ja 2,5 ml:11a etikkahappoanhydridia.Amino acids were sequentially coupled to the resin using a 2.0-3.0-fold molar excess of each protected amino acid as well as DIC. The resin was treated during subsequent coupling cycles with 0.269 g of Boc-Pro-OH, 0.610 g of Boc-Bth-OH, 0.311 g of Boc-Leu-OH-Cl 2, 0.375 g : 11a Boc-D-Tyr-OH, 0.610 g: 11a Boc-Bth-OH, 0.380 g: 11a Boc-Ser (benzyl) -OH,; 0.320 g of Boc-D-Pal (3) -OH, 0.390 g of Boc-D-pCl-Phe-OH, 0.400 g of Boc-D-Nal (2) -OH and 2.5 ml of acetic anhydride.
Hartsi poistettiin reaktioastiasta, pestiin C^C^: 35 Ha ja kuivattiin alipaineessa, jolloin saatiin 1,43 g » ·The resin was removed from the reaction vessel, washed with CH 2 Cl 2 and dried under reduced pressure to give 1.43 g.
IIII
92326 37 suojattua polypeptidi-hartsiyhdistelmaa. Peptidista poistettiin suojaukset, ja se irrotettiin hartsista ka-sittelemalla yhdistelmaå 25 ml :11a vedetdnta nestemais-ta HF:. 2,5 ml:n anisolia (akseptori) låsna ollessa Kel-5 F-reaktioastiassa 0°C:ssa 1 tunti, HF haihdutettiin ali- paineessa, jaljelle jaanyt N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal (3)-Ser-hArg(CG2CF3)2-D-Tyr-Leu-hArg(CH2CF3)2~Pro-D-Ala-NH2 (HF-suolanaan) pestiin eetterilla. JaSnnos uutettiin sitten jååetikalla (3 x 30 ml). Etikkahappouuttoliuos 10 haihdutettiin kuiviin. Puhdistamaton aine muunnettiin asetaattisuolaksi laskemalla se vedessa asetaattimuotoon muunnetun AG3-pylvaan (heikosti emaksinen tertiaarinen amiinihartsi lHpi. Eluaatin kylmakuivaus tuotti tulok-seksi 0,5 g puhdistamatonta peptidin asetaattisuolaa 15 valkoisena kiinteana aineena.92326 37 protected polypeptide-resin combination. The peptide was deprotected and removed from the resin by treatment with a combination of 25 mL of aqueous liquid HF. With 2.5 ml of anisole (acceptor) in a Kel-5 F reaction vessel at 0 ° C for 1 hour, the HF was evaporated under reduced pressure to leave N-Ac-D-Nal (2) -D-pCl-Phe -D-Pal (3) -Ser-hArg (CG2CF3) 2-D-Tyr-Leu-hArg (CH2CF3) 2-Pro-D-Ala-NH2 (as its HF salt) was washed with ether. The portion was then extracted with glacial acetic acid (3 x 30 mL). The acetic acid extraction solution 10 was evaporated to dryness. The crude material was converted to the acetate salt by applying it in water to an acetate-converted AG3 column (weakly basic tertiary amine resin 1 Hpi. Freeze-drying of the eluate gave 0.5 g of crude peptide acetate salt as a white solid.
Epapuhdas peptidi puhdistettiin HPLC:n avulla Licroprep RP-18-pylvaassa (25,40^um), jonka koko oli 2,5 x 100 cm ja joka oli saatettu tasapainoon 0,1 % CF3C02:ta CH2CN-H20-seoksessa (1:1) sisaltavålla pusku-20 rilla (pH 2,5). UV-valoa absorboiva (280 nm) paahuippu, joka eluoitui siina vaiheessa, kun pylvaMsta oli saatu noin 2 kertaa pylvS^n tilavuus eluaattia, kerattiin talteen ja haihdutettiin kuiviin seka kylmakuivattiin kolmesti tislatusta vedesta, jolloin saatiin 64 mg puh-25 dasta N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-hArg(CG2CF3)2- D-Tyr-Leu-hArg(CH2CF3)2-Pro-D-Ala-NH2:ta, foCj^5 = -17,96° (c = 0,4, HOAc).The crude peptide was purified by HPLC on a 2.5 x 100 cm Licroprep RP-18 column (25.40 μm) equilibrated with 0.1% CF 3 CO 2 in CH 2 CN-H 2 O (1: 1) with a buffer of 20 (pH 2.5). The UV-absorbing (280 nm) overhead peak, which eluted at about 2 times the column volume of eluate, was collected and evaporated to dryness and freeze-dried three times from distilled water to give 64 mg of pure N-Ac. -D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-hArg (CG2CF3) 2- D-Tyr-Leu-hArg (CH2CF3) 2-Pro-D-Ala-NH2 δ FCl 3 = -17.96 ° (c = 0.4, HOAc).
B. Menettelemalla samalla tavalla mutta kaytta-målla mainittujen aminohappojen tilalla asianmukaista .30 A-, B-, C-, D-, E-, F-, G- ja J-aminohappoa valmistet- tiin vastaavat dekapeptidit, joista on annettu esimerk-keja alia: 38 9/326 N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Pal(3)-Leu-Den-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Mbh-D-Pal(3)-Leu-Mbh-Pro-D-AlaNH2; 5 N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Pal(3)- D-Pal(3)-Leu-Bth-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Arg-D-Pal(3)-Leu-Deb-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Pal(3)- 10 L9u-3tn-Pro-D-AlaNH2; N-Ac-D-Nal (2)-D-pCl-Pne-D-Pal(3)-Ser-Tyr-D-Pal(3)-Leu-Mbh-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Pbe-D-Pal (3)-Ser-Tyr-D-Pal(3)-Leu-Pha-Pro-D-AlaNH2; 15 N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Trp-B. By proceeding in the same manner but using the appropriate amino acids in place of the 30 amino acids A, B, C, D, E, F, G and J, the corresponding decapeptides were prepared as exemplified. keja alia: 38 9/326 N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Tyr-D-Pal (3) -Leu-Den-Pro-D- AlaNH2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Mbh-D-Pal (3) -Leu-Mbh-Pro-D-AlaNH 2; 5 N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Pal (3) -D-Pal (3) -Leu-Bth-Pro-D-AlaNH2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Arg-D-Pal (3) -Leu-Deb-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Tyr-D-Pal (3) -10 L9u-3tn-Pro-D-AlaNH2; N-Ac-D-Nal (2) -D-pCl-Pne-D-Pal (3) -Ser-Tyr-D-Pal (3) -Leu-Mbh-Pro-D-AlaNH2; N-Ac-D-Nal (2) -D-pCl-Pbe-D-Pal (3) -Ser-Tyr-D-Pal (3) -Leu-Pha-Pro-D-AlaNH2; 15 N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Tyr-D-Trp-
Leu-3th-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Arg-D-Trp-Ls'J-Deh-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Arg-D-Trp-20 Leu-Bth-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-T rp-Ser-Arg-D-Trp-Leu-Pna-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Arg-D-Pal(3)-Leu-Pna-Pro-D-AlaNH2; *25 N-Ac-D-Nal(2)-D-pCl-Phe-D-T rp-Ser-Tyr-D-Btn-3th-Leu-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Arg-D-Trp-Ls'J-Deh-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Arg-D-Trp-20 Leu-Bth-Pro-D-AlaNH2; N-Ac-D-Nal (2) -D-pCl-Phe-D-T rp-Ser-Arg-D-Trp-Leu-Pna-Pro-D-AlaNH2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Arg-D-Pal (3) -Leu-PNA-Pro-D-AlaNH 2; * 25 N-Ac-D-Nal (2) -D-pCl-Phe-D-T rp-Ser-Tyr-D-Btn-
Leu-Bth-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Pna-Leu-Pha-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Deh-;.30 Leu-Den-Prb-D-AlaNH2; and : N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-3th-Leu-Bth-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Tyr-D-PNA-Leu-Pha-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Tyr-D-Deh-; 30 Leu-Den-Prb-D-AlaNH2; and: N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Tyr-D-3th-
Leu-Bth-Pro-D-AlaNH^.Leu-Bth-Pro-D-AlaNH ^.
C. Menettelemållå samalla tavalla mutta korvaamalla 35 mainitut aminohapot asianmukaisella A-, B-, C-, D-, E-, . F-, G- ja J-aminohapolla saadaan valmistetuksi vastaa- vat dekapeptidit, joista on annettu esimerkkeja alla: li 92326 39 N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Trp-Leu-Deh-Pro-D-AlaNH^; N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Trp-Leu-MPh-Pro-D-AlaNH^; 5 N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Trp-C. By proceeding in the same manner but replacing the 35 said amino acids with the appropriate A-, B-, C-, D-, E-,. The corresponding decapeptides are prepared with amino acids F, G and J, examples of which are given below: li 92326 39 N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Tyr- D-Trp-Leu-Deh-Pro-D-AlaNH ^; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Tyr-D-Trp-Leu-MPH-Pro-D-AlaNH ^; 5 N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Tyr-D-Trp-
Leu-Pha-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Den -Leu-Deh-Pro-D-AlaNh^; N-Ac-D-Nal (2)-D-pCl-Pne-D-Trp-Ser-Tyr-D-Mbn-Leu-Moh-Prc-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Mbh-Leu-Mbh-Prc-D-Ala NH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Pha-^ Leu-Pha-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Pbe-D-Trp-Ser-Arg-D-Trp-Leu-Nbh-Prc-D-Ala NH2; N-Ac-D-Nal(2) -D-pCl-Phe-D-Pal (3)-Ser-Arg-D-Pal(3)-Leu-Bth-Prc-D-AlaNH2; 2o N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Arg- D-Pal(3)-Leu-Mbn-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Arg-D-Tyr-Leu-Pha-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Tyr-D-Den-Leu-Deh-Pro-D-AlaNh 2; N-Ac-D-Nal (2) -D-pCl-Pne-D-Trp-Ser-Tyr-D-Mbn-Leu-Moh-Prc-D-AlaNH2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Tyr-D-Mbh-Leu-Mbh-Prc-D-Ala NH2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Tyr-D-Pha- ^ Leu-Pha-Pro-D-AlaNH2; N-Ac-D-Nal (2) -D-pCl-Pbe-D-Trp-Ser-Arg-D-Trp-Leu-Nbh-Prc-D-Ala NH2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Arg-D-Pal (3) -Leu-Bth-Prc-D-AlaNH2; 2o N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Arg-D-Pal (3) -Leu-Mbn-Pro-D-AlaNH2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Arg-D-Tyr
Leu-Den-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Arg-D-Tyr-25 Leu-Bth-Pro-D-AlaNH2; : N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Arg-D-Tyr-Den-Leu-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Arg-D-Tyr-25 Leu-Bth-Pro-D-AlaNH2; : N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Arg-D-Tyr-
Leu-Mbh-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Arg-D-Tyr-Le u-Ph a -Pr o-D- Ala NH2; 30 N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Arg-D-Tyr-Leu-Mbh-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Arg-D-Tyr-Le u-Ph a -Pr o-D-Ala NH2; 30 N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Arg-D-Tyr-
Leu-Bth-Prc-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Arg-D-Tyr-Leu-Mbh-Prc-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Arg-D-Tyr-35 Leu-Deh-Pro-D-AlaNH2; q o 3 9 6Leu-Bth-Prc-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Arg-D-Tyr-Leu-Mbh-Prc-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Arg-D-Tyr-35 Leu-Deh-Pro-D-AlaNH2; q o 3 9 6
40 st-OLO40 hours
N-Ac-D-Nal (2)-D-pCl-Phe-D-Pal(3)-Ser-Arg-D-Tyr-Leu-Pha-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Deh-D-Tyr-Leu-Deh-Pro-D-AlaNH2; 5 N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Mbh-D-Tyr-Leu-N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Arg-D-Tyr-Leu-Pha-Pro-D-AlaNH2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Deh-D-Tyr-Leu-Deh-Pro-D-AlaNH 2; 5 N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Mbh-D-Tyr-Leu-
Mbh-Pro-D-AlaNH9; N_Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-3tn-D-Tyr-Lej- 3tn-Pro-D-AlaNH2; N -Ac-D-Na 1 (2) -D-pCl-Pne-D-Trp-Ser-Pna-D-Ty r-Leu- 10Mbh-Pro-D-AlaNH9; N_Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-3tn-D-Tyr-Lej-3tn-Pro-D-AlaNH2; N -Ac-D-Na 1 (2) -D-pCl-Pne-D-Trp-Ser-Pna-D-Ty r-Leu- 10
Pha-Pro-D-AlaNH^; N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Deh-D-T rp-Leu-Deh-Pro-D-AlaNH^; N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Mbh-D-Trp-Leu-1 Mbn-Pro-D-AlaNH^; N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-3th-D-Trp-Leu-Pha-Pro-D-AlaNH ^; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Deh-D-T rp-Leu-Deh-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Mbh-D-Trp-Leu-1 Mbn-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-3th-D-Trp-Leu-
Btn-Pro-D-AlaNH^; N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Pha-D-T rp-Leu-Pha-Pro-D-AlaNH^; 2Q N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Deh-D-Pal(3)-Btn-Pro-D-AlaNH ^; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Pha-D-T rp-Leu-Pha-Pro-D-AlaNH 2; 2Q N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Deh-D-Pal (3) -
Leu-Deh-Pro-D-AlaNH^; N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Mbh-D-Pal(3)-Leu-Deh-Pro-D-AlaNH ^; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Mbh-D-Pal (3) -
Leu-Mbh-Pro-D-AlaNH2;Leu-Mbh-Pro-D-AlaNH 2;
N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-3tn-D-Pal(3)-«25 Leu-Bth-Pro-D-AlaNh^JN-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-3tn-D-Pal (3) - «25 Leu-Bth-Pro-D-AlaNh ^ J
N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Pha-D-Pal(3)-Leu-Pha-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Deh-D-Nal(2)-Leu-Deh-Pro-D-AlaNH2*j 2q N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Moh-D-Nal(2)- *: Leu-Mbh-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Pbe-D-Trp-Ser-3th-D-Nal(2)-Leu-3th-Pro-D-AlaNH2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Pha-D-Nal(2)-35 Leu-Pha-Pro-D-AlaNH2; li 41 52326 N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Pal(3)-D-Pal(3)-Leu-Deh-Pro-D-AlaNH2; N_Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Pal(3)-D-Pal(3)-Leu-Bth-Pro-D-AlaNH2; 5 N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Pal(3)- D-Pal(3)-Leu-Pha-Pro-D-AlaNH2; N_Ac-D-Nal(2)-0-pCl-Phe-D-Pal(3)-Ser-Pal(3)- 0-3al(3)-Leu-Mpn-Pro-D-AlaNH2.N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Pha-D-Pal (3) -Leu-Pha-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Deh-D-Nal (2) -Leu-Deh-Pro-D-AlaNH2 * j 2q N-Ac-D- Nal (2) -D-pCl-Phe-D-Trp-Ser-Moh-D-Nal (2) - *: Leu-Mbh-Pro-D-AlaNH2; N-Ac-D-Nal (2) -D-pCl-PBE-D-Trp-Ser-3th-D-Nal (2) -Leu-3th-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Pha-D-Nal (2) -35 Leu-Pha-Pro-D-AlaNH2; li 41 52326 N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Pal (3) -D-Pal (3) -Leu-Deh-Pro-D-AlaNH2 ; N_Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Pal (3) -D-Pal (3) -Leu-Bth-Pro-D-AlaNH 2; 5 N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Pal (3) -D-Pal (3) -Leu-Pha-Pro-D-AlaNH2; N_Ac-D-Nal (2) -O-pCl-Phe-D-Pal (3) -Ser-Pal (3) -0-3al (3) -Leu-Mpn-Pro-D-AlaNH2.
Esimerkki 2 10 N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-hArg(CH2CF3)2~ D-Tyr-Leu-hArg(CH2CF2)2~Pro-NHet C-terminaalisen Pro-NHCH2CH2:n sisåltavien analogien syntetisoinnissa kSytettiin esimerkissa 1 kuvatun synteesiohjelman kanssa identtistå ohjelmaa. Beckman 990-15 syntetisointilaitteen reaktioastiaan laitettiin 0,71 gExample 2 N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-hArg (CH2CF3) 2 ~ D-Tyr-Leu-hArg (CH2CF2) 2 ~ Pro-NHet In the synthesis of analogs containing C-terminal Pro-NHCH2CH2, a program identical to the synthesis program described in Example 1 was used. 0.71 g was placed in the reaction vessel of a Beckman 990-15 synthesizer
Boc-Pro-O-hartsia, joka oli valmistettu antamalla 1,3 ker-taisen modisen ylimaarSn kuivaa Boc-Pro-OH:n cesiumsuo-laa reagoida 1 % divinyylibentseenia sisaltavån kloori-metyylipolystyreenihartsin (Lab Systems, Inc.) kanssa.Boc-Pro-O resin prepared by reacting a 1.3-fold moderate excess of the dry cesium salt of Boc-Pro-OH with a chloromethylpolystyrene resin containing 1% divinylbenzene (Lab Systems, Inc.).
20 Kaytetty maara Boc-Pro-O-hartsia sisalsi 0,5 mmol proliinia.The amount of Boc-Pro-O resin used contained 0.5 mmol of proline.
Hartsiin liitettiin aminohappoja peråkkain kayttaen 2,0-3,0-kertaista moolista ylim&aråa kutakin suojattua aminohappoa seka DIC:ta. Hartsin annettiin siis reagoida perakkaisten liitantSsyklien aikana : '25 0,610 g:n kanssa Boc-Bth-OH:ta, 0,311 g:n kanssa Boc-Leu-0H*H20:ta, 0,375 g:n kanssa Boc-D-Tyr-OH:ta, 0,610 g:n kanssa Boc-Bth-OH:ta, 0,380 g:n kanssa Boc-Ser(bentsyyli)-OH:ta, .:30 0,320 g:n kanssa Boc-D-Pal (3)-OH:ta, 0,390 g:n kanssa Boc-D-pCl-Phe-OH:ta, 0,400 ml:n kanssa Boc-D-Nal(2)-OH:ta ja 2.5 ml:n kanssa etikkahappoanhydridia.Amino acids were sequentially coupled to the resin using a 2.0-3.0-fold molar excess of each protected amino acid and DIC. The resin was thus allowed to react during successive addition cycles: '25 with 0.610 g of Boc-Bth-OH, with 0.311 g of Boc-Leu-OH * H 2 O, with 0.375 g of Boc-D-Tyr-OH with 0.610 g of Boc-Bth-OH, with 0.380 g of Boc-Ser (benzyl) -OH, with 0.320 g of Boc-D-Pal (3) -OH with 0.390 g of Boc-D-pCl-Phe-OH, with 0.400 ml of Boc-D-Nal (2) -OH and with 2.5 ml of acetic anhydride.
Hartsi poistettiin reaktioastiasta, pestiin 35 CH2Cl2:lla ja kuivattiin alipaineessa, jolloin saatiin 1.5 g suojattua polypeptidi-hartsiyhdistelmSa.The resin was removed from the reaction vessel, washed with CH 2 Cl 2 and dried under reduced pressure to give 1.5 g of the protected polypeptide-resin combination.
92326 42 lyysillå, joka toteutettiin 25 ml :11a etyyliamiinia 2°C: ssa reaktioajan ollessa 18 tuntia. Etyyliamiinin annet-tiin haihtua, ja hartsi uutettiin metanolilla. Metanoli haihdutettiin, 2,5 ml:n kanssa anisolia ja 25 ml:n kans-5 sa uudelleen tislattua (CoF^sta) vedetontå nestemaista HF:a Kel-F-reaktioastiassa 0°C:ssa 1 tunti. HF haihdutettiin alipaineessa, ja jaånnos pestiin eetterilla. Jåannos liuotettiin 2 M etikkahappoon ja muunnettiin asetaattisuo-laksi laskemalla se vedessa asetaattimuotoon muunnetun 10 AC3-pylvaan lapi. Eluaatin kylmakuivaus tuotti tulokseksi 0,5 g puhdistamatonta peptidin asetaattisuolaa valkoisena kiinteana aineena. Lopullinen puhdistus toteutettiin HPLC-pylvaassa, jonka koko oli 2,5 x 100 mm ja joka sisalsi 40-50^um:n oktadekyylisilyloitua silikaa (Merck, Lichro-15 prep C-^g), kayttaen eluenttina 50-%:sta CHgCN:a (0,1 % CFgC02H:ta). Tuotteen kylmakuivaus vedesta tuotti tulokseksi 70 mg N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-hArg-(CH2CF2-D-Tyr-Leu-hArg(CH2CF3)2-ProNHEt:tå valkoisena jauheena.92326 by lysis in 25 ml of ethylamine at 2 ° C for a reaction time of 18 hours. The ethylamine was allowed to evaporate and the resin was extracted with methanol. The methanol was evaporated, with 2.5 ml of anisole and 25 ml of redistilled (from CoF) anhydrous liquid HF in a Kel-F reaction vessel at 0 ° C for 1 hour. The HF was evaporated under reduced pressure and the residue was washed with ether. The residue was dissolved in 2 M acetic acid and converted to the acetate salt by passing it through a pad of 10 AC3 columns converted to the acetate form in water. Freeze-drying of the eluate gave 0.5 g of the crude peptide acetate salt as a white solid. Final purification was performed on an HPLC column 2.5 x 100 mm in size containing 40-50 octadecylsilylated silica (Merck, Lichro-15 prep C-g) using 50% CH 2 CN as eluent. a (0.1% CF 2 CO 2 H). Freeze-drying of the product from water gave 70 mg of N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-hArg- (CH2CF2-D-Tyr-Leu-hArg (CH2CF3) 2- ProNHEt as a white powder.
20 Menettelemålla samalla tavalla mutta kayttamalla vaadittavia suojattuja aminohappoja saadaan valmistetuksi seuraavat yhdisteet: N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Pal(3)-Leu-Bth-Pro-NHEt, *;25 N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Pal(3)-Leu-By proceeding in the same manner but using the required protected amino acids, the following compounds are prepared: N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Tyr-D-Pal (3) -Leu -Bth-Pro-NHEt, *; 25 N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Tyr-D-Pal (3) -Leu-
Mbh-Pro-NHEt, N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Pal(3)-Leu-Pha-Pro-NHEt, N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Pal(3)-Leu-30 Deh-Pro-NHEt, ·' N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Arg-D-Nal(2)-Leu- 3th-Pro-NHEt.Mbh-Pro-NHEt, N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Tyr-D-Pal (3) -Leu-Pha-Pro-NHEt, N -Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Tyr-D-Pal (3) -Leu-30 Deh-Pro-NHEt, · 'N-Ac-D -Nal (2) -D-pCl-Phe-D-Trp-Ser-Arg-D-Nal (2) -Leu- 3th-Pro-NHEt.
Toistettaessa edellM esitetty lohkaisu ja korvat-^ taessa etyyliamiini stokiometrisella maaralla metyyli- _ ·« 43 Q07 o £ s <L 0 tai propyyliamiinia saadaan vastaavia edella mainittujen nonapeptidien metyyli- tai propyyliamideja„By repeating the above cleavage and replacing the ethylamine with a stoichiometric amount of methyl or propylamine, the corresponding methyl or propyl amides of the above-mentioned nonapeptides are obtained.
Esimerkki 3Example 3
Yhdisteita, jotka vastaavat kaavaa (I), voidaan 5 valmistaa myds klassisella liuossynteesilla.Compounds of formula (I) may be prepared by myds classical solution synthesis.
Esimerkiksi kahden pentapeptidifragmentin yhteen liittamisessa voidaan kayttaa seuraavaa menettelytapaa: N-Ac-D-Nal(2)-D-p-Cl-Phe- Boc-Den-Leu-Den-Pro-D-Ala-NH2 10 Q-?al(3)-Ser-Tyr-OMs (1) (2) — . v — H-D-Deh-Leu-Deh-Pro-D-Ala-Ny 'f N-Ac-D-Nal(2)-D-p-Cl-Phe-15 D-Pal(3)-Ser-Tyr-N3For example, the following procedure can be used to join two pentapeptide fragments: N-Ac-D-Nal (2) -Dp-Cl-Phe-Boc-Den-Leu-Den-Pro-D-Ala-NH2 10 Q-? Al (3) -Ser-Tyr-OMs (1) (2) -. v - H-D-Deh-Leu-Deh-Pro-D-Ala-Ny 'f N-Ac-D-Nal (2) -D-p-Cl-Phe-15 D-Pal (3) -Ser-Tyr-N3
VV
H-D-Deh-H-D-deH
Leu-Deh-Pro-AzaGlyNH2 (3) <r N-Ac-D-Nal(2)-D-p-Cl-Phe-D-Pal(3)-Ser-Tyr-D-Deh-Leu-Oeh-Pro-D-Ala-NH? 20 ^Leu-Deh-Pro-AzaGlyNH2 (3) -r N-Ac-D-Nal (2) -Dp-Cl-Phe-D-Pal (3) -Ser-Tyr-D-Deh-Leu-Oeh-Pro- D-Ala-NH? 20 ^
Erillisten fragmenttien liittaminen yhteen voidaan toteuttaa asyyliatsidimenetelmalla (J. Honzel et al.,Joining of individual fragments can be accomplished by the acylazide method (J. Honzel et al.,
Coll. Czech. Chem. Commun. 26 (1971) 2333), DCC/HBT-lii-*.25 tannalla, difenyylifosforyyliatsidiliitannalla tai jolla- kin muulla rasemisoimattomalla fragmenttien liitantatek-niikalla. Yhdiste (2) voidaan valmistaa edella esitetylla tavalla, ja yhdiste (1) voidaan valmistaa esimerkissa 1 kuvatun kaltaisilla menetelmilla. Yhdiste (3) valmis-30 tetaan yhdisteesta (2) poistamalla Cbz hydraamalla, mita « ί seuraa N-Boc-N,N*-guanidinodietyyli-D-homoarginiinin liit taminen DCC-HBT-yhdistelmaa tai jotakin muuta, alalia tun-nettua liitantaainetta kayttaen.Coll. Czech. Chem. Commun. 26 (1971) 2333), a DCC / HBT ligand, a diphenylphosphoryl azide coupling, or some other non-racemic fragment coupling technique. Compound (2) can be prepared as described above, and compound (1) can be prepared by methods similar to those described in Example 1. Compound (3) is prepared from compound (2) by removing Cbz by hydrogenation, followed by the addition of N-Boc-N, N * -guanidinodiethyl-D-homoarginine to a DCC-HBT combination or other excipient known in the art. using.
Tallå tavalla yhteen liitettavat fragmentit ovat 35 usein peptideja tai aminohappoja. Vaihtoehtoisesti N-ter- 92326 44 minaalinen nonapeptidi voidaan valmistaa kiinteåfaasi-tai liuosmenetelmalla ja liittaa sen jålkeen alkyyliamii-niin tai semikarbatsidihydrokloridiin disykloheksyyli-karbodi-imidin ja hydroksibentsotriatsolin avulla tai 5 jollakin muulla liitåntåmenetelmållå, jolloin saadaan vastaavan kaltaisia yhdisteita.The fragments to be joined in this way are often peptides or amino acids. Alternatively, the N-tert-92326 44 terminal nonapeptide may be prepared by a solid phase or solution method and then coupled to an alkylamine or semicarbazide hydrochloride by dicyclohexylcarbodiimide and hydroxybenzotriazole or by any other coupling method.
Esimerkki 4Example 4
Suolojen valmistus A. Liuos, joka sisalsi 0,1 g (N-Ac-D-Nal(2)x, 10 D-pCl-Phe2, D-Pal(3)3,8, Btn8, D-Ala"^) LHRH :n vetyfluori-disuolaa (ts esimerkki 1) liuotettuna 50 ml:aan vettå, laskettiin pylvaan lapi, joka sisalsi 50 g Dowex 3-anio-ninvaihtohartsia, joka oli saatettu tasapainoon aikai-semmin etikkahapolla ja pesty deionisoidulla vedellå.Preparation of salts A. Solution containing 0.1 g (N-Ac-D-Nal (2) x, 10 D-pCl-Phe2, D-Pal (3) 3.8, Btn8, D-Ala ") The hydrogen fluoride disalt salt of LHRH (i.e. Example 1) dissolved in 50 ml of water was counted on a column containing 50 g of Dowex 3-anion exchange resin previously equilibrated with acetic acid and washed with deionized water.
15 Pylvas eluoitiin deionisoidulla vedellå ja eluaatti kylmå-kuivattiin, jolloin saatiin vastaavaa (N-Ac-D-Nal(2)^, 9 O C O T Λ D-pCl-Phe“, D-Pal(3) ' , Bth , D-Ala )LHRH:n etikkahappo- suolaa.The column was eluted with deionized water and the eluate was freeze-dried to give the corresponding N-Ac-D-Nal (2) ^, 9 OCOT Λ D-pCl-Phe “, D-Pal (3) ', Bth, D-Ala ) Acetic acid salt of LHRH.
Toistettaessa edellå esitetty menettely mutta kor-20 vattaessa etikkahappo muilla hapoilla hartsin tasapaino-tuksessa voidaan valmistaa esimerkiksi vastaavia suola-hapon, bromivetyhapon, rikkihapon, fosforihapon, typpi-hapon, bentsoehapon sekå muiden samankaltaisten happojen suoloja.By repeating the above procedure but replacing acetic acid with other acids in equilibrating the resin, for example, corresponding salts of hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, benzoic acid and the like can be prepared.
·* 25 Vastaavasti voidaan valmistaa muita LHRH:n kanssa analogisten peptidien, joita tåsså selityksesså on esitetty, happoadditiosuoloja.· * 25 Similarly, other acid addition salts of the peptides analogous to LHRH disclosed in this specification can be prepared.
B. Suoloja, jolden vesiliukoisuus on heikko, voidaan valmistaa vedestå saostamalla haluttua happoa hyvåk- , 30 si kåyttåen. Esimerkiksi: » « ·· Sinkkitannaattisuola - Liuos, joka sisålsi 10 mg (N-Ac-D-B. Salts with low water solubility can be prepared from water by precipitation using the desired acid. For example: »« ·· Zinc tannate salt - Solution containing 10 mg (N-Ac-D-
Nal(2)1, D-pCl-Phe2, D-Pal(3)3,6, Bth8, D-Ala10)LHRHrn etikkahapposuolaa 0,1 mlrssa vettå, kåsiteltiin liuok-sella, joka sisålsi 8 mg parkkihappoa 0,08 ml:ssa 0,25 M 35 NaOH:ta. LHRH-analogin liuokseen lisåttiin vålittomåsti « _ · 92326 45 liuos, joka sisalsi 5 mg ZnSO^-7H20:ta 0,1 ml:ssa vettå.Nal (2) 1, D-pCl-Phe2, D-Pal (3) 3,6, Bth8, D-Ala10) acetic acid salt of LHRH in 0.1 ml of water was treated with a solution containing 8 mg of tannic acid in 0.08 ml in 0.25 M 35 NaOH. A solution of 5 mg of ZnSO 4 -7H 2 O in 0.1 mL of water was immediately added to the solution of the LHRH analog.
Tulokseksi saatu suspensio laimennettiin 1 ml :11a vettå, ja sakka erotettiin sentrifugoimalla. Supernatant-ti poistettiin dekantoimalla, ja jåannos pestiin kahdesti 5 1 ml:n erillå vettå sentrifugoimalla sakka ja dekantoi malla supernatantti. Sakka kuivattiin alipaineessa, jol-loin saatiin 15 mg edellå mainitun LHRH-analogin sinkki-tannaattisekasuolaa.The resulting suspension was diluted with 1 ml of water, and the precipitate was separated by centrifugation. The supernatant was removed by decantation, and the residue was washed twice with 5 ml of separate water by centrifugation of the precipitate and decantation of the supernatant. The precipitate was dried under reduced pressure to give 15 mg of the zinc tannate mixed salt of the above-mentioned LHRH analog.
Pamoaattisuola — Liuokseen, joka sisålsi 50 mg 10 (N-Ac-D-Nal(2)1, D-pCl-Phe2, D-Pal(3)3'6, Bth8, D-Ala10)-LHRH:n etikkahapposuolaa seoksessa, joka sisålsi 1,6 ml etanolia ja 0,1 ml 0,25 M NaOH:ta, lisåttiin liuos, joka sisålsi 11 mg pamonihappoa 0,3 ml:ssa 0,25 M NaOH:ta. Liuottimet poistettiin alennetussa paineessa, jåånnos 15 suspendoitiin 2 ml:aan vettå, suspensio sentrifugoitiin, ja supernatantti dekantoitiin. Sakka pestiin 1,5 ml :11a vettå ja sentrifugoitiin, ja supernatantti dekantoitiin. Sakka kuivattiin alipaineessa, jolloin saatiin 54 mg edellå mainitun LHRH-analogin pamoaattisuolaa.Pamoate salt - To a solution containing 50 mg of the acetic acid salt of 10 (N-Ac-D-Nal (2) 1, D-pCl-Phe2, D-Pal (3) 3'6, Bth8, D-Ala10) -LHRH in a mixture , containing 1.6 ml of ethanol and 0.1 ml of 0.25 M NaOH, a solution of 11 mg of pamonic acid in 0.3 ml of 0.25 M NaOH was added. The solvents were removed under reduced pressure, the residue was suspended in 2 ml of water, the suspension was centrifuged, and the supernatant was decanted. The precipitate was washed with 1.5 ml of water and centrifuged, and the supernatant was decanted. The precipitate was dried under reduced pressure to give 54 mg of the pamoate salt of the above-mentioned LHRH analog.
20 Samalla tavalla voidaan valmistaa muitakin suoloja, joiden vesiliukoisuus on heikko.20 Similarly, other salts with poor water solubility can be prepared.
C. Happoadditiosuolan valmistus vapaasta pepti-diståC. Preparation of acid addition salt from free peptide
Liuokseen joka sisålsi 50 mg (N-Ac-D-Nal(2) ^, . 25 D-pCl-Phe2, D-Pal(3)3,6, Bth8, D-Ala10)LHRH:ta vapaana emåksenå, lisåttiin 30 ml 1 N etikkahappoa. Tulokseksi saatu liuos kylmåkuivattiin, jolloin saatiin 50 mg edellå mainitun LHRH-analogin etikkahapposuolaa.To a solution of 50 mg (N-Ac-D-Nal (2) 2, D-pCl-Phe 2, D-Pal (3) 3,6, Bth 8, D-Ala 10) LHRH as a free base was added 30 ml of 1 N acetic acid. The resulting solution was lyophilized to give 50 mg of the acetic acid salt of the above-mentioned LHRH analog.
Vastaavasti korvattaessa etikkahappo muilla hapoil-30 la (stokiometrisesti ekvivalenttisina måårinå suhteessa peptidiin) saadaan muita tåsså selityksesså esitettyjen peptidien happoadditiosuoloja, esimerkiksi suolahapon, bromivetyhapon, rikkihapon, fosforihapon ja typpihapon suoloja.Similarly, substitution of acetic acid with other acids (in stoichiometrically equivalent amounts relative to the peptide) provides other acid addition salts of the peptides disclosed herein, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid and nitric acid.
35 D. Metallikationin muodostaman suolan, esimerkik si sinkkisuolan, valmistus : Liuokseen, joka sisålsi 50 mg (N-Ac-D-Nal(2) 46 92326 D-pCl-Phe2, D-Pal(3)3'6, Bth8, D-Ala10)LHRH:n etikkahap-posuolaa seoksessa, joka sisalsi 0,4 ml 0,25 M NaOH:ta, 0,3 ml vetta ja 1 ml etanolia, lisattiin liuos, joka si-5 salsi 15 mg ZnSO^·71^0:ta 0,2 ml:ssa vetta. Sakka sentri-fugoitiin ja supernatantti erotettiin dekantoimalla. Sakka pestiin 1 ml:11a vetta sentrifugoimalla ja dekantoimalla supernatantti. Sakka kuivattiin alipaineessa, jol-loin saatiin edelia mainitun LHRH-analogin sinkkisuolaa.35 D. Preparation of a salt formed by a metal cation, for example a zinc salt: To a solution containing 50 mg of N-Ac-D-Nal (2) 46 92326 D-pCl-Phe2, D-Pal (3) 3'6, Bth8, D-Ala10) Acetic acid salt of LHRH in a mixture of 0.4 ml of 0.25 M NaOH, 0.3 ml of water and 1 ml of ethanol was added to a solution of 15 mg of ZnSO 4. ^ 0 in 0.2 ml of water. The precipitate was centrifuged and the supernatant was separated by decantation. The precipitate was washed with 1 ml of water by centrifugation and decantation of the supernatant. The precipitate was dried under reduced pressure to give the above zinc salt of said LHRH analog.
10 Samalla tavalla voidaan valmistaa suoloja muiden moniarvoisten kationien, esimerkiksi kalsiumin, vismutin, bariumin, magnesiumin, alumiinin, kuparin, koboltin, nik-kelin, kadmiumin ja muiden samankaltaisten, kanssa.Salts can be prepared in a similar manner with other polyvalent cations, for example, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium and the like.
Esimerkki 5 15 Suolojen muuntaminen vapaaksi emMkseksiExample 5 Conversion of salts to free emM
Liuos, joka sisalsi 50 mg (N-Ac-D-Nal(2)*, D-pCl-Phe2, D-Pal(3)3'6, Bth8, D-Ala10)LHRH:n etikkahap-posuolaa 25 ml:ssa vetta laskettiin pylvaan lapi, joka sisalsi 50 g Dowex l:ta (voimakkaasti emaksinen ammonium- 20 anioninvaihtohartsi), joka oli tasapainotettu NaOH-liuok- sella hydroksidivastaionin aikaansaamiseksi. Pylvas elu-oitiin 150 ml:11a vetta, ja eluaatti kylmakuivattiin, jolloin saatiin 45 mg vastaavaa polypeptidia vapaana emaksena.Solution containing 50 mg of acetic acid salt of LHRH (N-Ac-D-Nal (2) *, D-pCl-Phe2, D-Pal (3) 3'6, Bth8, D-Ala10) in 25 ml: water was lowered onto a column containing 50 g of Dowex 1 (a strongly basic ammonium anion exchange resin) equilibrated with NaOH solution to give a hydroxide counterion. The column was eluted with 150 ml of water, and the eluate was lyophilized to give 45 mg of the corresponding polypeptide as a free mother.
25 Muut tassa selityksessa esitettyjen peptidiyhdis- teiden happoadditiosuolat voidaan muuntaa vapaiksi emak-siksi vastaavalla tavalla.Other acid addition salts of the peptide compounds disclosed herein may be converted to the free sow in a similar manner.
KarakterisointitiedotCharacterizing
Edelia olevien esimerkkien mukaisesti valmistettu- 30 jen yhdisteiden karakterisointitiedot on esitetty taulu-koissa 1 ja 2: 92326 47 S D iZ<Z<<Z<< ~Ζ<<<Ζ*<Ζ<<<<ί<Ζ<<< ν' - Q “ O x x 5 - * < c x i x Q -. -. * -. -. * -. . - L« * - * - * * m — C x. * _' . o r— ^ ® κτ ΓΝ o m . CM <N o u o ~ o ^ O* o O 6 o “ o o o' 6 O o o o o o o o o' o o O o ° ο°ο°ουθου °°οουυοοουοοοοοουου awCJ·*— Ow-W*-'^·'^-' 00-^-2^2^2^0^0^0^^ O O O o 9 o 9 · ®The characterization data of the compounds prepared according to the previous examples are shown in Tables 1 and 2: 92326 47 SD iZ <Z << Z << ~ Ζ <<< Ζ * <Ζ <<<< ί <Ζ <<< ν '- Q “O xx 5 - * <cxix Q -. -. * -. -. * -. . - L «* - * - * * m - C x. * _ '. o r— ^ ® κτ ΓΝ o m. CM <N ouo ~ o ^ O * o O 6 o “ooo '6 O oooooooo' oo O o ° ο ° ο ° ουθου °° οουυοοουοοοοοουο awCJ · * - Ow-W * - '^ ·' ^ - '00- ^ -2 ^ 2 ^ 2 ^ 0 ^ 0 ^ 0 ^^ OOO o 9 o 9 · ®
OwOw99000 ^ w ® « _— ιΠ psj — U3 vO ® ® ® ^S'SSSSSfNO'T^SSaOO^^ N - O o IT1 ^ 1 y, , , . .. . .OwOw99000 ^ w ® «_— ιΠ psj - U3 vO ® ® ® ^ S'SSSSSfNO'T ^ SSaOO ^^ N - O o IT1 ^ 1 y,,,. ... .
I (ΝθΟΓ~Γ^<Νθι°*ί~ — — ηί — ηίίηΚ®ηίη!«»!η — ^I (ΝθΟΓ ~ Γ ^ <Νθι ° * ί ~ - - ηί - ηίίηΚ®ηίη! «»! Η - ^
L -fM----(NM- CN — — ^^ Τ,, Γι i I I I I I I IL -fM ---- (NM- CN - - ^^ Τ ,, Γι i I I I I I I I
0 i i i I i i i I I .......i i i I i.....0 i i i I i i i I I ....... i i i I i .....
E-HE-H
!l sssk;5;ss s;si;s:s;ss!s;s;;s li3 ÉéåÉÉÉÉÉÉ ÉÉåÉoÉÉÉÉÉÉÉÉÉÉÉÉÉ * * * * * *! l sssk; 5; ss s; si; s: s; ss! s; s ;; s li3 YYYYMMDD YYYYYYYYYYYYYYYYYYY * * * * * *
K XK X
x j£ X J x ¥ Xx j £ X J x ¥ X
¥^ ϊο £ S ¥=^¥x _l — x_l— XXX -J <=> -J g¥ ^ ϊο £ S ¥ = ^ ¥ x _l - x_l— XXX -J <=> -J g
o" — ¥o" — — — ¥ X X «Γ OO _| Xo "- ¥ o" - - - ¥ X X «Γ OO _ | X
X X— O _»—<©©_I O' I Qi — — — — QJX X— O _ »- <© acting _I O 'I Qi - - - - QJ
x gx c — ~ « I--== X XX β < ίο O ^ Xx gx c - ~ «I - == X XX β <ίο O ^ X
x x — < o— q«oo-)XX-j — i — — x -j _i x _j < i — < - — — g _i — < CI < O g — — Κ~ΙΟ X OlOO<<OOX— O I - I — xo fs) O TO Q .XXXX— Q N I I--_ _l O — o O Q < -J — — _J— *ΟΟΩ:ΧίΕα:<»'",*ΩΩ< % K> · I X © * X — O CD CM X _J X X I CD POU. , I -O « “ . . X® Λ® O 2° * frt • Q£ © -— * λ j — j i η ^ ^03 ¢0 Q ^ X X X ^ CM—*· » ^ ^ .xx - <o— q «oo-) XX-j - i - - x -j _i x _j <i - <- - - g _i - <CI <O g - - Κ ~ ΙΟ X OlOO << OOX— OI - I - xo fs) O TO Q .XXXX— QNI I --_ _l O - o OQ <-J - - _J— * ΟΟΩ: ΧίΕα: <»'", * ΩΩ <% K> · IX © * X - O CD CM X _J XXI CD POU., I -O «“.. X® Λ® O 2 ° * frt • Q £ © -— * λ j - ji η ^ ^ 03 ¢ 0 Q ^ XXX ^ CM— * · »^ ^.
< X— < m in — o — — . (NO n« . I C < I g g X —X (NOO _l O I -P<X— <m in - o - -. (NO n «. I C <I g g X —X (NOO _l O I -P
I _l O I INIlO - o o s -4- Tm ~ 1N0B o — I O X X _J 3 O +- — — C tø O — — O — O — O— — — LU x ΓΊ +- CN . < O _l _l — O — LU «Ό < * λ , -O < - ΚΛ T\l C — <3 Ο η I U -O · -03 — — O — σ> — +-— ' ® U>I _l O I INIlO - o o s -4- Tm ~ 1N0B o - I O X X _J 3 O + - - - C tø O - - O - O - O— - - LU x ΓΊ + - CN. <O _l _l - O - LU «Ό <* λ, -O <- ΚΛ T \ l C - <3 Ο η I U -O · -03 - - O - σ> - + -— '® U>
i—I i— — ifflu.x— < — — ·*-σ>— o—mmo® k-o o — muoi®0® — Mi — I i— - ifflu.x— <- - · * -σ> - o — mmo® k-o o - muoi®0® - M
® o o — o o < i < < lu L. σι w σι u_ —-___· " * l < l » - > ♦; ~® o o - o o <i <<lu L. σι w σι u_ —-___ · "* l <l» -> ♦; ~
_ O — · <N ΤΊ — I O I I — < U O) L U m® - N««-<r<OI<®lll P_ O - · <N ΤΊ - I O I I - <U O) L U m® - N «« - <r <OI <®lll P
O Q.<®-*-XoiQ.Oooi£<U<'',U-'nniX — — < £ · Γ L I ‘ · i—1 U1 «I * LJ f 1 i -CD . » L. .£^£XO — XP^^ * -® - ^ O ® m r-(O Q. <® - * - XoiQ.Oooi £ <U <'', U-'nniX - - <£ · Γ L I '· i — 1 U1 «I * LJ f 1 i -CD. »L.. £ ^ £ XO - XP ^^ * -® - ^ O ® m r- (
75 o O in — — < ® Kto æ < O .2 . o ni m O — IIOO w 2„·-ΐι. P75 o O in - - <® Kto æ <O .2. o ni m O - IIOO w 2 „· -ΐι. P
Ai ~ ·» Oln2n- - n2-o .o C. X X — σι a o -oo m (J nj 3 cmo — i_ c. » (m (m -- . <n —. ® — σ> O O σ> u - -® — <n y m — +- r. jjAi ~ · »Oln2n- - n2-o .o C. XX - σι ao -oo m (J nj 3 cmo - i_ c.» (M (m -. <N -. ® - σ> OO σ> u - -® - <nym - + - r. Jj
.3 f k\3<<it 4-T3 m® 4-— mm£ — —u<æ® m σ x < ~ inui x -P.3 f k \ 3 << it 4-T3 m® 4-— mm £ - —u <æ® m σ x <~ Inui x -P
·~2 u-æ£i-iuOjiu.-u ooKr-'-'-ao l ui;'!0 3 — m — - -m — ——'QfN—ajniiuSuu® -m— ni— — >. — luo>-^ m Z σι x σιιη « w σισιστΝ-*- σι·ΡΧ~· »<< ·<η -*-ox tu σιο. ζ “ i Ρ1 zf.· ~ 2 u-æ £ i-iuOjiu.-u ooKr -'-'- ao l ui; '! 0 3 - m - - -m - ——' QfN — ajniiuSuu® -m— ni— ->. - luo> - ^ m Z σι x σιιη «w σισιστΝ - * - σι · ΡΧ ~ ·» << · <η - * - ox tu σιο. i “i Ρ1 zf.
•ο ΐ-οι-σίν. _ lllxiuluo o»m -c ^ m aiiiaouLvx»<L 1) -εθ)£<ι-ηΧ££^σΐ£<σι<».ιηιη ι_< σισσ£^ >.>~< i -c “i• ο ΐ-οι-σίν. _ lllxiuluo o »m -c ^ m aiiiaouLvx» <L 1) -εθ) £ <ι-ηΧ ££ ^ σΐ £ <σι <». ιηιη ι_ <σισσ £ ^>.> ~ <i -c“ i
I I- - - I« - - ·β” I Ϊ Lf< »»< - L. L_ L. I I _I _I —I - MI I- - - I «- - · β” I Ϊ Lf <»» <- L. L_ L. I I _I _I —I - M
O <® <0 Q Ό lø®® i. < Ο I < I Ό I, L. -Ό < < < O o » · * oO <® <0 Q Ό lø®® i. <Ο I <I Ό I, L. -Ό <<<O o »· * o
' m immmmf-immSim.i .m . . » m « - »m m · - - « m i—I'm immmmf-immSim.i .m. . »M« - »m m · - -« m i — I
— ό — — — — ^ — λ ^c^ λ — m η n —®®»n— ®®® — kl kl m <—<- ό - - - - ^ - λ ^ c ^ λ - m η n —®® »n— ®®® - kl kl m <- <
Kl .Κ1ΚΙΚΙΚΙΙΊΚΙΚΙ .Kl- .-KI · - .C - Kl Kl - - - ; ^ f.Kl .Κ1ΚΙΚΙΚΙΙΊΚΙΚΙ .Kl-.-KI · - .C - Kl Kl - - -; ^ f.
® — k> wwwwwwwm»n w «κι m—mmin „ _ m κι κι κί — +J® - k> wwwwwwwm »n w« κι m — mmin „_ m κι κι κί - + J
4J — Q. — — __ — — — O.Q. — wo.— — O.Q. — “0.0.0. — — ww — w— ^u m ol.ooooOool.uo — u — ou.^ — euui-oo — — — — o rn4J - Q. - - __ - - - O.Q. - wo.— - O.Q. - “0.0.0. - - ww - w— ^ u m ol.ooooOool.uo - u - ou. ^ - euui-oo - - - - o rn
, 0-1— Itilllti-t· O. Ot— OQ.I— h * I H l· ►“Q.Q.OOO^Q- W, 0-1— Itilllti-t · O. Ot— OQ.I— h * I H l · ► “Q.Q.OOO ^ Q- W
Ή I I I 'liTT Ιιιιη.ιη.11 ·0-Ι I I 1110.0.0-4-1. >-Ή I I I 'liTT Ιιιιη.ιη.11 · 0-Ι I I 1110.0.0-4-1. > -
IT-J QQQQqqQQQqqqiqiqQQ IQQQQqq i tlTO CIT-J QQQQqqQQQqqqiqiqQQ IQQQQqq i tlTO C
ZL -“--..----..Q -Q ..-0------Q000 - K-1 JU (VNNNNNNNINNNN .<N .(Μ ΓΜ ΓΜ -m fSCMINnifM ... -tN ,—|ZL - “--..---- .. Q -Q ..- 0 ------ Q000 - K-1 JU (VNNNNNNNINNNN. <N. (Μ ΓΜ ΓΜ -m fSCMINnifM ... -tN , - |
Sh ΦΦΦΦφφΟΦΦΟΑΟηΐ φ(Ν φ φ Φ fN ΦΦΦΦφφίΝΓΝ ΟΙ. Μ φ — . — ££££££££££££¢^¢^££¢££££££¢¢¢9-0 <0Sh ΦΦΦΦφφΟΦΦΟΑΟηΐ φ (Ν φ φ Φ fN ΦΦΦΦφφίΝΓΝ ΟΙ. Μ φ -. - ££££££££££££ ¢ ^ ¢ ^ ££ ¢ ££££££ ¢¢ 9-0 <0
οοοΰοοόοοΰοοι!.οι!.οθΡι*-ΡΡ00ΰθα-ιί^υΐυθ Oοοοΰοοόοοΰοοι! .οι! .οθΡι * -ΡΡ00ΰθα-ιί ^ υΐυθ O
p-p-p-f-p-p-p-p-p-f-p-p-p-p-p-p-p-p-p-p-f-p-f-p-p-p-p-p-p·?· g.p-p-p-f-p-p-p-p-p-f-p-p-p-p-p-p-p-p-p-p-f-p-f-p-p-p-p-p-p ·? · g.
QOQQqqOQCI 3QCiQQQ©0Q0QOQOqq0qQQQ Q*i CMCMrMfNrMCMCMCMCNcMrMCMrMCNCMCNf^r^CNCNfNCNCNrsir^CNfMf^fNf^ q — — ϋ — rx — — — — — — — — — — — — — — — — — — — cQOQQqqOQCI 3QCiQQQ © 0Q0QOQOqq0qQQQ Q * i CMCMrMfNrMCMCMCMCNcMrMCMrMCNCMCNf ^ r - CN - c - - - -
CQQOqqOQ OOeoeeec<0<jO«3C <3O<Jt0<0H)«3CCCQQOqqOQ OOeoeeec <0 <jO «3C <3O <Jt0 <0H)« 3CC
?????????????????????????????? « UOUUgnUUOoguuUUOUOUUOoUouOu^U^ η ιι·ΐιιΐιΐιιιιιιιΐιιιιιΐιιιι·'' h;?????????????????????????????? «UOUUgnUUOoguuUUOUOUUOoUouOu ^ U ^ η ιι · ΐιιΐιΐιιιιιιιΐιιιιιιιιι '' 'h;
X zzzzzzzzzzzzzzzzzzzzzzzzzzzzzz MX zzzzzzzzzzzzzzzzzzzzzzzzzzzzz M
H — — — — — — — — — — — JjH - - - - - - - - - - - Jj
C (tJC (tJ
I i rn ^^♦«C^miOOO^^OO'inOOOfN^^vCOO' - — opm — cmI i rn ^^ ♦ «C ^ miOOO ^^ OO'inOOOfN ^^ vCOO '- - opm - cm
Xi Ocd — η-(Ν— c^ — Ov^r-ooD — fMfMOCOCMCMOmm — comoQ^ 1ΝΐΛΝ\£)-ιΛ^^®Λ^^ΓΜ0ίΜ-ί"Ιί'ρ,*0'ΰΛα>ΐΛΡΒ'0" || Ό * o -i-^iOi^i«i=|x|x|r|-i|!£|x|s|2i|g|^|a|s:|s|K|s|?:|s|R|s|p;|«|^|^|Si| 48 o O 7 9 £Xi Ocd - η- (Ν— c ^ - Ov ^ r-ooD - fMfMOCOCMCMOmm - comoQ ^ 1ΝΐΛΝ \ £) -ιΛ ^^ ®Λ ^^ ΓΜ0ίΜ-ί "Ιί'ρ, * 0'ΰΛα> ΐΛΡΒ'0" || Ό * o -i- ^ iOi ^ i «i = | x | x | r | -i |! £ | x | s | 2i | g | ^ | a | s: | s | K | s |?: | s | R | s | w, | «| ^ | ^ | Si | 48 o O 7 9 £
W L. VW L. V
Taulukko 2 Aminohappoanalyysit 15 (RS-26334): Ser 0,96 (1); Pro 0,96 (1); Ala 0,98 (1); Leu 0,95 (1); Tyr 1,07 (1); Lys 5 0,09 (0); NH3 1,82 (1); Trp 2,12 (2); p-Cl-Phe 1.00 (1); hrtrg(CH2CF3 )2 0,89 (1); Nal(2 ) 1,09 (1).Table 2 Amino acid analyzes 15 (RS-26334): Ser 0.96 (1); Pro 0.96 (1); Ala 0.98 (1); Leu 0.95 (1); Tyr 1.07 (1); Lys 5 0.09 (0); NH3 1.82 (1); Trp 2.12 (2); p-Cl-Phe 1.00 (1); hrtrg (CH 2 CF 3) 2 0.89 (1); Nal (2) 1.09 (1).
21 (RS-26126): Ser 1,01 (1); Pro 0,99 (1); Ala 0,98 i n — (1); Leu 0,95 (1); Lys 0,20 (1); NH3 1,27 (1); Arg 1,08 (1); Pal(3) 2,10 (2); p-Cl-Phe 1,11 (1); hArg(CH2CF3) 0,79 (1); Nal(2) 1,02 (1).21 (RS-26126): Ser 1.01 (1); Pro 0.99 (1); Ala 0.98 and n - (1); Leu 0.95 (1); Lys 0.20 (1); NH3 1.27 (1); Arg 1.08 (1); Pal (3) 2.10 (2); p-Cl-Phe 1.11 (1); hArg (CH 2 CF 3) 0.79 (1); Nal (2) 1.02 (1).
15 2_2 (RS-26906): Ser 1,03 (1); Pro 0,97 (1); Ala 0,98 (1); Leu 0,90 (1); NH3 2,25 (1); Trp 2.30 (2); Arg 1,12 (1); p-Cl-Phe 1,03 (1); hArg(CH2CF3)2 0,99 (1);15 2_2 (RS-26906): Ser 1.03 (1); Pro 0.97 (1); Ala 0.98 (1); Leu 0.90 (1); NH3 2.25 (1); Trp 2.30 (2); Arg 1.12 (1); p-Cl-Phe 1.03 (1); hArg (CH 2 CF 3) 2 0.99 (1);
Nal(2) 1,12 (1).Nal (2) 1.12 (1).
20 r 31 (RS-64736): Ser 0,84 (1); Pro 0,89 (1); Ala 0,99 (1); Leu 1,01 (1); Lys 1,05 (1); ΝΗ? 0,91 (1); hArg(Et2) 1,04 (1); Pal(3) ; 25 2,42 (2); p-F-Phe 1,21 (1); Nal(2) 1,13 (1).20 r 31 (RS-64736): Ser 0.84 (1); Pro 0.89 (1); Ala 0.99 (1); Leu 1.01 (1); Lys 1.05 (1); ΝΗ? 0.91 (1); hArg (Et2) 1.04 (1); Pal (3); 25 2.42 (2); p-F-Phe 1.21 (1); Nal (2) 1.13 (1).
32 (RS-64802): Ser 0,92 (1); Pro 0,95 (1); Ala 1,01 (1); Leu 0,99 (1); Lys 1,01 (1); NH3 30 0,96 (1); hArg(Et2) 1,02 (1); Pal(3) 2.30 (2); p-F-Phe 1,15 (1); Nal(2) 0,96 (1).32 (RS-64802): Ser 0.92 (1); Pro 0.95 (1); Area 1.01 (1); Leu 0.99 (1); Lys 1.01 (1); NH 3 0.96 (1); hArg (Et2) 1.02 (1); Pal (3) 2.30 (2); p-F-Phe 1.15 (1); Nal (2) 0.96 (1).
39 (RS-12972): Ser 0,76 (1); Pro 1,06 (1); Ala 0,89 (1); Leu 1,04 (1) ; Tyr 1,07 (1); 35 p-Cl-Phe 1,17 (1); NH3 1,03 (1);39 (RS-12972): Ser 0.76 (1); Pro 1.06 (1); Ala 0.89 (1); Leu 1.04 (1); Tyr 1.07 (1); 35 p-Cl-Phe 1.17 (1); NH3 1.03 (1);
Pal(3 ) 2,10 (2); hArg(C2H5> . : CH2CF3) 1,66 (1); Nal(2) 1,14 (1).Pal (3) 2.10 (2); hArg (C 2 H 5 O 2: CH 2 CF 3) 1.66 (1); Nal (2) 1.14 (1).
IIII
, .'· "7 S,. '· "7 S
49 yοζο49 yοζο
Yhdisteiden bioloqinen aktiivisuus (a) Testosteronitasojen aleneminen Testosteronitasojen aleneminen mitattiin seuraavaa testausmenettelya kayttaen: 5 Suoritettiin kaksi tutkimusta kayttaen toisessa 11 ja toisessa 13 taysikasvuista, urospuolista, intaktia be-aglekoiraa (iåltSSn 1,5-6,5 vuotta, toimittaja Syntex, LRE, Ridglan tai White Eagle). Kullakin viikolla 6 viikon ajan elaimet jaettiin sattumanvaraisesti johonkin 11 tai 10 13 annosryhmasta (apuainetta tai erilaisia annoksia ku- takin LHRH:n antagonistia) niin, etta kuhunkin ryhmaan tuli 6 koiraa. Ryhmaan 1 kuuluneet saivat yhtena ihon-alaisena ruiskeena 0,1 ml apuainetta ruumiinpaino-kg:a kohden. Muihin ryhmiin kuuluneet elaimet saivat jaljem-15 påna esitetyssa luettelossa 1 esitetyt annokset (^ug yh-distetta/0,1 ml:n ruisketilavuus/ruumiinpaino-kg). Apu-aineena oli propyleeniglykolin ja fysiologisen suolaliu-oksen seos suhteessa 1:1. Kultakin koiralta otettiin påan valtimon kautta juuri ennen ruiskeen antamista ja 30 mi-20 nuuttia sekS 1, 2, 4, 6, 8 ja 24 tuntia ruiskeen annon jSl-keen noin 3 ml:n hepariinipitoinen verinåyte. Verinaytteen sijoitettiin valittfimSsti ottamisen jålkeen jaihin. Plasma erotettiin sentrifugoimalla ja imusuodattamalla 2 tun-. nin kuluessa ja sailytettiin -20°C:ssa ennen testoste- 25 ronitasojen RIA-mååritysta.Biological Activity of Compounds (a) Decreased Testosterone Levels Decreased testosterone levels were measured using the following test procedure: 5 Two studies were performed in one of 11 and 13 full-grown, male, intact be-agle dogs (iåltSSn 1.5-6.5 years, supplied by Syntex or White Eagle). For 6 weeks each week, the animals were randomly assigned to one of 11 or 10 13 dose groups (excipient or different doses of each LHRH antagonist) so that 6 dogs were included in each group. Group 1 patients received a single subcutaneous injection of 0.1 ml of excipient per kg of body weight. Animals in the other groups received the doses shown in List 1 below (μg compound / 0.1 ml injection volume / kg body weight). The excipient was a 1: 1 mixture of propylene glycol and physiological saline. A blood sample of approximately 3 ml of heparin was taken from each dog through the artery just prior to injection and 30 ml to 20 minutes sec 1, 2, 4, 6, 8 and 24 hours after injection into the injection. The blood sample was randomly placed in Jai after collection. Plasma was separated by centrifugation and suction filtration for 2 hours. and stored at -20 ° C prior to RIA determination of testosterone levels.
Testosteronitasot mitattiin radioimmunologisella maarityksella. Naiden kahden tukimuksen tulokset yhdis-tettiin, ja piirrettiin testosteronitasoja annostasojen funktiona kuvaava kayra ID^Q-arvojen (annos, joka vaadi-‘30 taan testosteronitason alentamiseksi 50 %:iin maksimiar- vostaan 8 tunnin aikana) saamiseksi. Nama arvot on esi-tetty taulukossa 3, jossa on esitetty myos teho suhteessa LHRH:n standardiantagonistiin Detirelix (RS-68439).Testosterone levels were measured by radioimmunoassay. The results of the two studies were combined, and testosterone levels plotted as a function of dose levels were obtained to obtain Kayra ID ^ Q values (dose required to reduce testosterone levels to 50% of their maximum value in 8 hours). These values are shown in Table 3, which also shows the potency against the standard LHRH antagonist Detirelix (RS-68439).
50 'J'/ 3 /! ^50 'J' / 3 /! ^
Luettelo 1 AnnosryhmåtList 1 Dose groups
Tutkimus 1 (11 annosryhmåa):Study 1 (11 dose groups):
Ryhmå Yhdiste (RS-nro) Annos 1 Valiaine 2 RS-26306 0,06yg/0,lml/kg 3 RS-26306 0,25yg/0,lml/kg 4 RS-26306 1,0yg/0, l.nl/kg 5 RS-26306 4 10yg/0,lul/kg 6 RS-26306 16,0y3/0,lml/kg 7 RS-26723 0f06yg/0,lml/kg 8 RS-26723 0;25yQ/0,lml/kg 9 RS-26723 1f0yg/0,lml/kg 10 RS-26723 4,Oyg/QfIml/kg 11 RS-26723 16;Oyg/ύ,lml/kgGroup Compound (RS No.) Dose 1 Element 2 RS-26306 0.06yg / 0.1ml / kg 3 RS-26306 0.25yg / 0.1ml / kg 4 RS-26306 1.0yg / 0.1l./l/ kg 5 RS-26306 4 10yg / 0, lul / kg 6 RS-26306 16.0y3 / 0.1 lml / kg 7 RS-26723 0f06yg / 0.1 lml / kg 8 RS-26723 0; 25yQ / 0, lml / kg 9 RS-26723 1f0yg / 0.1ml / kg 10 RS-26723 4, Oyg / QfIml / kg 11 RS-26723 16; Oyg / ύ.1ml / kg
Tutkimus 2 (13 annosryhmaa)Study 2 (13 dose groups)
Ryhma Yhdiste (RS-nro) Annos 1 valiaine 2 RS-68439 (standard!) 0,25 ug/0,lml/kg 3 RS-68439 (standard!) 1,0 ug/0 imi/kg 4 RS-68439 (standard!) 4,0 ug/0,lml/kg : 5 RS-68439 (standard!) 16,0 ug/0,lml/kg 6 RS-15378 0,25 ug/0,lml/kg 7 RS-15378 1,0 ug/0,lmi/kg 8 RS-15378 4,Q yg/0 lml/kg 9 RS-15378 16,0 ug/0,lml/kg 10 RS-15678 °»2S pg/O.lml/kg .1' 11 RS-15678 1,0 Ug/0 lml/kg 12 RS-15678 4,0 ug/0,lml/kg 13 RS-15i78 ug/ojlml/kg 92326 51 C Λ α> ^ c 00 5 ι ^ ^ ^ '2 Ο Ο <3* CNJ CO C0 Ε ·*· ·> *» ·*» ** Ο) Ο <-Η Ο —I ΙΑ Γ- C -Η ·*— ν— —^ ^ -— m -ΡGroup Compound (RS No.) Dose 1 Element 2 RS-68439 (standard!) 0.25 ug / 0.1 ml / kg 3 RS-68439 (standard!) 1.0 ug / 0 imi / kg 4 RS-68439 ( standard!) 4.0 ug / 0.1 ml / kg: 5 RS-68439 (standard!) 16.0 ug / 0.1 ml / kg 6 RS-15378 0.25 ug / 0.1 ml / kg 7 RS-15378 1.0 μg / 0.1 lmi / kg δ RS-15378 4, γ yg / 0 lml / kg 9 RS-15378 16.0 μg / 0.1 lml / kg 10 RS-15678 ° »2S pg / 0.1l / kg .1 '11 RS-15678 1.0 Ug / 0 lml / kg 12 RS-15678 4.0 ug / 0.1 lml / kg 13 RS-15i78 ug / ojlml / kg 92326 51 C Λ α> ^ c 00 5 ι ^ ^ ^ '2 Ο Ο <3 * CNJ CO C0 Ε · * · ·> * »· *» ** Ο) Ο <-Η Ο —I ΙΑ Γ- C -Η · * - ν— - ^ ^ -— m -Ρ
Jl ·η σι er. οι σι σ· c 5J -9 .X.*.*-*: χ. a <0_·Γ-| \ \ S. \ \ .JH.Jl · η σι er. οι σι σ · c 5J -9 .X. *. * - *: χ. a <0_ · Γ- | \ \ S. \ \ .JH.
c “ I ss ss s s •?l| m - r^m σ. ^ K\ i: UM-rH^ rsi (Λ _i o (—) ΠΙΟ -H CO _ (C O O Æ 00 4J ^ oS 2 roc “I ss ss s s •? l | m - r ^ m σ. ^ K \ i: UM-rH ^ rsi (Λ _i o (-) ΠΙΟ -H CO _ (C O O Æ 00 4J ^ oS 2 ro
•η tn -P Pj ϋ 4J• η tn -P Pj ϋ 4J
C o m tn SC o m tn S
O u 0) o X SO u 0) o X S
i_i o m +J o JSi_i o m + J o JS
Q) w LA 5Q) w LA 5
p J-> C S 9 -Hp J-> C S 9 -H
0) tu 0) H H E0) tu 0) H H E
0 tn to m Ρ Μ X ^ ^ Λ i2 tn o 3 co iA a-' -.—- g <D E -P i tn * · —< £ -P -H 3 -Η I V w w ^ 1 +j ·η C q 3 di -c -d0 tn to m Ρ Μ X ^ ^ Λ i2 tn o 3 co iA a- '-.—- g <DE -P i tn * · - <£ -P -H 3 -Η IV ww ^ 1 + j · η C q 3 di -c -d
_ <P ® C O \ r -c -C -C a O_ <P ® C O \ r -c -C -C a O
O^^OCiiVD m « PH si > qStnrH® Ό ® Λ « •h 3 tn o) > -p -h .* ro aO ^^ OCiiVD m «PH si> qStnrH® Ό ® Λ« • h 3 tn o)> -p -h. * Ro a
tn r-t fH Qtn r-t fH Q
C 2a!Sfl5p gC 2a! Sfl5p g
iB^US-S SiB ^ US-S S
3 ^ 4J Λ 8 ^ 2 'O Ό Ό VO c 5 *H —( —i g in i i i i i Ti3 ^ 4J Λ 8 ^ 2 'O Ό Ό VO c 5 * H - (—i g in i i i i i Ti
•H Ifl I +J -H• H Ifl I + J -H
>CtnQ)‘£' vo miA vo *d H 8 S ϋ J2 1 ° °1 “ 5 i! .5 L 8 ° ° ° ° o ;§ y u-hc5^> CtnQ) ‘£’ vo miA vo * d H 8 S ϋ J2 1 ° ° 1 “5 i! .5 L 8 ° ° ° ° o; § y u-hc5 ^
m ae 5 $ 3 J? Sm ae 5 $ 3 J? S
3 § § M 3 Cn VO “ Ό "" xw 0) -Ρ O ·Ρ -P 3 « «* .. J3 " J S5 5J> «· "L "L -L ϊ t ; J5 S T C — 3 o" <" ' C X ^H -H i—i i—t e n P ή o; ro _ ro ro ro ro O £ ro T CL X Q- q_~h<t} 3* Q_ —I i or i i ci jj ro -7 ' · '—> Q X Q qi-h§ 3 § M 3 Cn VO “Ό" "xw 0) -Ρ O · Ρ -P 3« «* .. J3" J S5 5J> «·" L "L -L ϊ t; J5 STC - 3 o" <"'CX ^ H -H i — ii — ten P ή o; ro _ ro ro ro ro O £ ro T CL X Q- q_ ~ h <t} 3 * Q_ —I i or ii ci jj ro -7 '·' -> QXQ Qi-h
4-* * t-J Q «_!_·> ·* Q TJ4- * * t-J Q «_! _ ·> · * Q TJ
C! ^ “ t· «OfM i—t CN Cvl ·. nj ro S N,. 5 N <υ o <υ X qj co {§ 10 n, ^5 JJ C turo jz cr n ^ >C! ^ “T ·« OfM i — t CN Cvl ·. nj ro S N ,. 5 N <υ o <υ X qj co {§ 10 n, ^ 5 JJ C Turo jz cr n ^>
. 5J mi-, x rj CL ro CL X Q. N. 5J mi-, x rj CL ro CL X Q. N
3 S Q- «i i^h i _ι I 4J ro ” 3 1 q (-C1 ri J. ^ et -} -HU 12 § jg 3 ^Sro * Ω ‘d σ .1 I d«* Ϊη" is :· ^_CN*'H"^cv»^“ef ή * -f n3 S Q- «ii ^ hi _ι I 4J ro” 3 1 q (-C1 ri J. ^ et -} -HU 12 § jg 3 ^ Sro * Ω 'd σ .1 I d «* Ϊη" is: · ^ _CN * 'H "^ cv» ^ “ef ή * -fn
*1 (0 mT^a JJ Q Λ _! C* 1 (0 mT ^ a JJ Q Λ _! C
' ·” s 3«* 3 S SS S«r- s«- S'· ”S 3« * 3 S SS S «r- s« - S
2 -5 S 2* "ro ^~Cvi ^ II2 -5 S 2 * "ro ^ ~ Cvi ^ II
O Ih zhh Zd ZdZ4-> z*j Λί O ω OVIUKV I X CO i r O lu VO I U oO Ih zhh Zd ZdZ4-> z * j Λί O ω OVIUKV I X CO i r O lu VO I U o
3+JS fAQwtNQ ΙΓ"0|Γ-ο!ϋ OQw IO3 + JS fAQwtNQ ΙΓ "0 | Γ-ο! Ϋ OQw IO
rt [ή H <r ID>r7t_J\0|_jKV I CT ΓΑ I r-T) Ort [ή H <r ID> r7t_J \ 0 | _jKV I CT ΓΑ I r-T) O
COOLiVOtj.iAtj.moM VO Λ U «Η VOddCvidlA H H c[ 5 CM <5 <5 3^i i ix i ισ»ι ισ>ι ιχ i i r- il roxw COZIOOZIhCOZU^ZI unzi EHXCi X^QX^dCC«—'dX1—>_J X< |Q * 92326 52 (b) Histamiinin vapautusvaikutus ja ovulaation vastainen vaikutusCOOLiVOtj.iAtj.moM VO Λ U «Η VOddCvidlA HH c [5 CM <5 <5 3 ^ ii ix i ισ» ι ισ> ι ιχ ii r- il roxw COZIOOZIhCOZU ^ ZI unzi EHXCi X ^ QX ^ dCC «- ' dX1 -> _ J X <| Q * 92326 52 (b) Histamine release effect and anti-ovulatory effect
Seuraavia menettelytapoja kåyttåen vertailtiin tyy-pillisten tåmån keksinnon mukaisten yhdisteiden histamii-5 ninvapautusvaikutusta ja ovulaation vastaista vaikutusta.Using the following procedures, the histamine-5 release and anti-ovulatory activity of typical compounds of this invention were compared.
1. Histamiinin vapautustutkimus1. Histamine release study
Kolmesta tai neljåstå urospuolisesta Sprague-Dawley-rotasta (350 g, Iffa-Credo, Ransa) eristettiin sekalaisia peritoneaalisia soluja antaen niille intraperitoneaalisena 10 ruiskeena 10 ml 0,9 %:sta NaCl:a (joka sisålsi 50^ug hepa-riinia/ml). Sen jålkeen kun vatsaonteloa on hierottu ke-vyesti 1 minuutin ajan, peritoneaaliset solut eristettiin ja yhdistettiin, ja niitå sentrifugoitiin 5 minuuttia no-peudella 1500 min Kolmesti Ca++-vapaalla Krebs-Ringer-15 puskurilla (KRB), jonka koostumus oli seuraava: 141,9 mM NaCl, 4,7 mM KCl, 1,2 mM MgS04, 2,5 mM Na2HP04, 0,6 raM KH2P04; huuhtelemisen jålkeen solut laskettiin mikroskoo-pin alla ja laimennettiin sopivalla tilavuusmåårållå Ca -vapa-ata KBR: tå niin, ettå saavutettiin solutiheydek-20 si noin 2 x 10® solua/ml. 0,3 ml:n eriå esilåmmitettiin 0,3 ml:n kanssa Ca++-vapaata KRB:tå 5 minuuttia 35°C:ssa, ja lisåttiin 0,1 ml asianmukaista testattavaa lååkeliuos-ta. 5 minuutin kuluttua kåynnistettiin reaktio lisååmållå 0,1 ml Ca -pitoista KRB:tå halutun pitoisuuden saavuttami-. 25 seksi. 15 minuutin kuluttua reaktio pysåytettiin lisååmållå 2,5 ml jååkylmåå Ca++-vapaata KRB:tå ja asettamalla suspensio jåihin. Solususpension sentrifugoinnin jålkeen mååritettiin supernatantin histamiinipitoisuus fluori-metrisesti menetelmållå, jonka ovat esittåneet Shore et al. 30 /Immunol. 127 (1959) 182-1867» jåttåen pois uuttoprosessi (eksitaatio aallonpituudella 365 nm, emissio aallonpituu-della 450 nm). Yksikåån reagensseista ei fluoresoinut 0-ftaalidialdehydin kanssa kokeissa kåytettyinå pitoisuuk-sina.Miscellaneous peritoneal cells were isolated from three or four male Sprague-Dawley rats (350 g, Iffa-Credo, Ransa) by intraperitoneal injection of 10 ml of 0.9% NaCl (containing 50 μg heparin / ml). ). After lightly rubbing the abdominal cavity for 1 minute, the peritoneal cells were isolated and pooled and centrifuged for 5 minutes at 1500 min. Three times with Ca ++ - free Krebs-Ringer-15 buffer (KRB) having the following composition: 141, 9 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO 4, 2.5 mM Na 2 HPO 4, 0.6 μM KH 2 PO 4; after rinsing, the cells were counted under a microscope and diluted with an appropriate volume of Ca-free KBR to achieve a cell density of about 2 x 10® cells / ml. A 0.3 ml aliquot was preheated with 0.3 ml of Ca ++ - free KRB for 5 minutes at 35 ° C, and 0.1 ml of the appropriate test drug solution was added. After 5 minutes, the reaction was started by adding 0.1 ml of Ca-containing KRB to reach the desired concentration. 25 sex. After 15 minutes, the reaction was quenched by the addition of 2.5 mL of ice-cold Ca ++ -free KRB and placing the suspension on them. After centrifugation of the cell suspension, the histamine content of the supernatant was determined fluorometrically by the method of Shore et al. 30 / Immunol. 127 (1959) 182-1867 »omitting the extraction process (excitation at 365 nm, emission at 450 nm). None of the reagents fluoresced with O-phthalaldialdehyde at the concentrations used in the experiments.
35 Solususpension kokonaishistamiinipitoisuus mååri- 53 9 2 3 2 6 tettiin ultraåånikåsittelyn (2 min, pulssitaajuus 5/s) jålkeen.The total histamine content of the cell suspension was determined after sonication (2 min, pulse rate 5 / s).
Kaikista mitatuista arvoista vahennettiin spontaani histamiinin vapautuminen.Spontaneous histamine release was subtracted from all measured values.
5 - Laskut5 - Invoices
Prosentuaalinen histamiinin vapautuminen lasket-tiin seuraavan yhtalSn avulla:Percent histamine release was calculated using the following equation:
Histamiinia soluista vapaassa supernatantissa -spontaani vapautuminen 10 - x 100Spontaneous release of histamine from cells in free supernatant 10 - x 100
Kokonaishistamiinimåarå solususpensiossa - Reagenssit ja laitteet: O-ftaalidialdehydi hankittiin Flukalta (nro 79760) . 15 Kaikki muut yhdisteet olivat analyysilaatua ja liuotet-tiin Ca++-vapaaseen KRB:hen. KSytetty fluorimetri oli Perkin Elmer 2000 -malli. UltraSSnikåsittely tehtiin VIBRA-CELL-laitteella (Sonics Materials) mikrokarkia kayttaen.Total histamine in cell suspension - Reagents and equipment: O-phthalic dialdehyde was purchased from Fluka (No. 79760). All other compounds were of analytical grade and dissolved in Ca ++ - free KRB. The fluorimeter used was a Perkin Elmer 2000 model. UltraSSn treatment was performed with a VIBRA-CELL (Sonics Materials) using a microcarrier.
20 2. Ovulaation vastaisuuden tutkiminen20 2. Investigation of anti-ovulation
Menettely: Naarasrottia sopeutetaan laboratorio-olosuhteisiin (valon ja pimean suhde 14:10, valot paalla klor 5 aamulla) vShintSan 7-10 vuorokautta. Kullekin ro-talle tehdSan pSivittåinen ematinhuuhtelu klo 7,30 - 9,00 25 ainakin 12 vuorokauden ajan. Émåtinhuuhtelunesteen sytolo-gia tutkitaan mikroskooppisesti estrussyklin vaiheen maå-rittåmiseksi. Rotat, joilla on ollut ainakin 2 normaalia nelipaivSistM syklia ennen parhaillaan olevaa syklia, valitaan. Odotetun estruspaivan aamuna rotat tapetaan, 30 munanjohtimet poistetaan, ja niistS tutkitaan vastairron-neiden munasolujen esiintyminen. Munasolut "houkutellaan" ulos munanjohtimesta ja lasketaan. Niiden naaraiden, joilla esiintyy ovulaatiota, prosentuaalinen m&årå esitetaSn graafisesti annoksen logaritmin funktiona ED^^-arvon 35 laskemiseksi ovulaation vastaiselle vaikutukselle.Procedure: Female rats are adapted to laboratory conditions (light to dark ratio 14:10, lights on chlorine 5 in the morning) vShintSan for 7-10 days. For each rat, perform a daily mother rinse from 7.30 to 9.00 for at least 12 days. The cytology of the rinsing fluid is examined microscopically to determine the phase of the estrus cycle. Rats that have had at least 2 normal four-day cycles before the current cycle are selected. On the morning of the expected day of estrus, the rats are sacrificed, the fallopian tubes are removed, and the presence of newly ejaculated oocytes is examined. The eggs are "attracted" out of the fallopian tube and counted. The percentage of females experiencing ovulation is plotted as a function of the logarithm of the dose to calculate an ED50 for the anti-ovulatory effect.
Histamiinin vapautusvaikutus ja ovulaation vastai-nen vaikutus on esitetty seuraavassa taulukossa 4.The histamine release effect and the anti-ovulatory effect are shown in Table 4 below.
54 92326 C tn ^—- 0 3 O' •Η 3 d ® ® ® S! y 3 (ΟιγΪγ-'ΟΟ^νΟΟιΛ ΟιΓ.Γ^σ'Ο'ιΟ V irt |M ·*> ^ *, 4' * ^ 1 ·.54 92326 C tn ^ —- 0 3 O '• Η 3 d ® ® ® S! y 3 (ΟιγΪγ-'ΟΟ ^ νΟΟιΛ ΟιΓ.Γ ^ σ'Ο'ιΟ V irt | M · *> ^ *, 4 '* ^ 1 ·.
(flto O o — o — — 0<N(V1000 00 i—I -P O O O — — til - 3 i/ι in j s 01 3 c i TI "d ^ CN κι ιβ ® ® t n C (θ' o o - - - « « 3 o- O* ° * - s 1 ti ^ +l + l + l + l + l + l 1ΛΟ— tt ® C 4Jmo od — m <e r» ° °i_ ^ ® c-·^ ® 0_ \o_ ^ 5 W gj in "l T. ov »6 m ί o ιτ m o æ O o o" ιίΓ - £ o — *H -HfpQ Oo cs in ® (N — — 10 -P S > W — (0(flto O o - o - - 0 <N (V1000 00 i — I -POOO - - til - 3 i / ι in js 01 3 ci TI "d ^ CN κι ιβ ® ® tn C (θ 'oo - - - «« 3 o- O * ° * - s 1 ti ^ + l + l + l + l + l + l 1ΛΟ— tt ® C 4Jmo od - m <er »° ° i_ ^ ® c- · ^ ® 0_ \ o_ ^ 5 W gj in "l T. ov» 6 m ί o ιτ mo æ O oo "ιίΓ - £ o - * H -HfpQ Oo cs in ® (N - - 10 -PS> W - (0
rOr O
i—i x 5 ϊ ii — i x 5 ϊ i
> X X _l X X> X X _l X X
0 ¥ ¥o i τ 5 si _ -J p — T £ J° JH, o" o' — — ¥ X X o' c ° S"c -» T2 d $ = Ϊ -c <0 ¥ ¥ o i τ 5 si _ -J p - T £ J ° JH, o "o '- - ¥ X X o' c ° S" c - »T2 d $ = Ϊ -c <
,-. T — — O «JO P X X P — I, -. T - - O «JO P X X P - I
5 xp< <* —- — α p — < o O o; — i _ x i ® < o © x — O i · r- To αΧχχο (ni — — p o — o oo ·0ίΧοί»-«-*ό^ζθ—— β « κ> <D x o CO ϊ ι χ ® kn . i — o «— OD ^ I) ΧχΟ — *_J — _J—»L/l© O < “ — < X cn5 xp <<* —- - α p - <o O o; - i _ xi ® <o © x - O i · r- To αΧχχο (ni - - po - o oo · 0ίΧοί »-« - * ό ^ ζθ—— β «κ> <D xo CO ϊ ι χ ® kn .i - o «- OD ^ I) ΧχΟ - * _J - _J—» L / l © O <“- <X cn
χ X — <—O — <NO ~ * i q < i o; —Xχ X - <—O - <NO ~ * i q <i o; -X
CO X _J C I (NO — © +· <N C<j0 O — I O X 3 0 r-t -J — — Q -V — C “ UJ I f <N * < Q » J CD w < — O <· K*\ «3 — CwOUJ I ·® — W O) •P O— I GO IL.—*< — 0)ww ΙΛ NQ® KO o L.CO X _J CI (NO - © + · <NC <j0 O - IOX 3 0 rt -J - - Q -V - C “UJ I f <N * <Q» J CD w <- O <· K * \ «3 - CwOUJ I · ® - WO) • PO— I GO IL .— * <- 0) ww ΙΛ NQ® KO o L.
r-j -·βθΛϋ<ι<ί®σιΐΐ(Λ ι<r-j - · βθΛϋ <ι <ί®σιΐΐ (Λ ι <
x o — « o* r* I O i < ^ t o OflD «-* <N O < «Cx o - «o * r * I O i <^ t o OflD« - * <N O <«C
(0 — < © -t-xo_. q2<< IN U n ni - j; Q. < I · UJ o *®_“· •c2xo~xo< .o x4 I Q iTv w —- æ ΚΌ0 vO · · O (N fNo « I © K\ (0 o.. » 819-- - -i® ® ~·χχ^σ>ο — ~(0 - <© -t-xo_. Q2 << IN U n ni - j; Q. <I · UJ o * ®_ “· • c2xo ~ xo <.o x4 IQ iTv w —- æ ΚΌ0 vO · · O (N fNo «I © K \ (0 o ..» 819-- - -i® ® ~ · χχ ^ σ> ο - ~
S «®__ — l. L. (M <M — CN ~ — 01(3 o Cj)p * — <NS «®__ - L. L. (M <M - CN ~ - 01 (3 o Cj) p * - <N
- \ΟΙ^>3<<·*-Χ3·*-— fNU''wl_<®=X- \ ΟΙ ^> 3 << · * -Χ3 · * -— fNU''wl_ <® = X
- ~3,ffl22iuueu3<-< pioi<£~aO- ~ 3, ffl22iuueu3 <- <pioi <£ ~ aO
r; CNNW *«^^w^C0uj£Ll£ *'Jw« p + x σι® ® οι σ> σι coi “ — »<< .® « o οι p ωοι.σιι.ι-ι.ΐ-ΐ.Ο' σι® j: £ ® σι tu ι_ l.r CNNW * «^^ w ^ C0uj £ Ll £ * 'Jw« p + x σι® ® οι σ> σι coi “-» << .® «o οι p ωοι.σιι.ι-ι.ΐ-ΐ.Ο 'σι® j: £ ® σι tu ι_ l.
C x-<l>><<<<i.loi · «aL-<< , eorKhirirKiL ® ® i_ < σι 2 -cC x- <l>> <<<< i.loi · «aL - <<, eorKhirirKiL ® ® i_ <σι 2 -c
p l-i_ « . I « *| £r<01D< »l-ι Ip l-i_ «. I «* | £ r <01D <»l-ι I
•fH <<®®qi0®®qI|®1-l.«®<OO• fH << ®®qi0®®qI | ®1-l. «® <OO
en\ ΐ £ * · · * * · % O q · < ^ Ό » £ « * w i i in in m «i in in in . «κι . « . «η .κι κι (Ti -p ΙΟ®®-«®-« — μ rn » · κνκνκν®κνκνκν — -.Kl . · rt. ΚΝ . ®® 01 -H a. q. _ _ — — ___ww — Q.O.W — a. — — i rr-i Ρι.σοβθοβο — _«ji_l — 5*-σ*> p iti »71-Q-Q.a.ti-Q.tLQ.eoa-i-i- c q. i- o. cl -d .d Α,'ι,Α,,|ίΐ-ο-ΑΑ,ο-ΧΑ,Α S_i a0000®Q00* i O O q i Ω Q q oen \ ΐ £ * · · * * ·% O q · <^ Ό »£« * w i i in in m «i in in. «Κι. «. «Η .κι κι (Ti -p ΙΟ®®-« ®- «- μ rn» · κνκνκν®κνκνκν - -.Kl. · Rt. ΚΝ. ®® 01 -H aq _ _ - - ___ww - QOW - a - - i rr-i Ρι.σοβθοβο - _ «ji_l - 5 * -σ *> p iti» 71-QQ.a.ti-Q.tLQ.eoa-ii- c q. I- o. Cl -d .d Α, 'ι, Α ,, | ίΐ-ο-ΑΑ, ο-ΧΑ, Α S_i a0000®Q00 * i OO Qi Ω Q qo
C (N. <N IN N Λ N N N « » . CN CN ON .CN CN CN CNC (N. <N IN N Λ N N N «». CN CN ON .CN CN CN CN
rt) ΦΦΦΦΦΦΦΦΦ CN CN φφφΓΝ φφφφ p Sefrr-seiFjF-si1rt) ΦΦΦΦΦΦΦΦΦ CN CN φφφΓΝ φφφφ p Sefrr-seiFjF-si1
Cn iii 1 iiiiil·11·!!*1!!!! * 03 OoOOOPOOO li- ιί. 00(3ι[θϋϋϋ ^ p Λ ? ? P- p-f p- p- p-f p- f1?· i" P· P- ? •o·-! 00000“000“000000000 drO . . ......Cn iii 1 iiiiil · 11 · !! * 1 !!!! * 03 OoOOOPOOO li- ιί. 00 (3ι [θϋϋϋ ^ p Λ?? P- p-f p- p- p-f p- f1? · I "P · P-? • o · -! 00000" 000 "000000000 drO.. ......
<ϋ 4J rt. Λ ,."3. -. rt. rt."A. -. rt. — rt. rt. «."A rt. rt."A<ϋ 4J rt. Λ,. "3. -. Rt. Rt." A. -. rt. - rt. rt. «." A rt. Rt. "A
I P N(NNCNCNN|N(N(NN(NIN(N(NINNCN(nINI P N (NNCNCNN | N (N (NN (NIN (N (NINNCN (nIN
ac o rrrrr-rrr-r--rr--r- CL· > ^«50<300ooO«J«jCJ«3c«3®®C<3ac o rrrrr-rrr-r - rr - r- CL ·> ^ «50 <300ooO« J «jCJ« 3c «3®®C <3
^ OugugUuguOoOUuuUUgU^ OugugUuguOoOUuuUUgU
•Hj3 <<<<<<<·<<<<<<<·<<<<< ij<n Α''|'Α'|,Α|ΑΑ'ιΑΑ·Α w +J ΖΖΖΖΖΖΪΖΖΖΖΖΖΖΖΖΖΖΖ •• Hj3 <<<<<<< · <<<<<<< <<<<< ij <n Α '' | 'Α' |, Α | ΑΑ'ιΑΑ · Α w + J ΖΖΖΖΖΖΪΖΖΖΖΖΖΖΖΖΖΖΖ •
• I• I
’ 0 rr p c 0 I σ'-»Ν·οοσ'®οο®σ;ο®®ΐΝΓ.®'τ — — i/ rn ®oo — γ.ον!Τ· — oio®cNmooocNn>i2— ® TT Xi 2®r»® — η η ® n cn cn - <5 — ®T;®Oi® Λ Pi ® — ®® — ®>0®® — ®®®ffllO®^^^ Z3 ®m — — io~cnvcn®cncncn^cncn®io®'0 rr pc 0 I σ' - »Ν · οοσ'®οο®σ; ο®®ΐΝΓ.®'τ - - i / rn ®oo - γ.ον! Τ · - oio®cNmooocNn> i2— ® TT Xi 2®r »® - η η ® n cn cn - <5 - ®T; ®Oi® Λ Pi ® - ®® - ®> 0®® - ®®®ffllO® ^^^ Z3 ®m - - io ~ ^ cnvcn®cncncn cncn®io®
i-II-i
I % cN|®|^|r.|Di|^|®|z|^|®|o|-|<N|®|,|®|®|S|o| H w 55 9 2 3 2 6I% cN | ® | ^ | r. | Di | ^ | ® | z | ^ | ® | o | - | <N | ® |, | ® | ® | S | o | H w 55 9 2 3 2 6
Yhdisteiden akuutti myrkyllisyysAcute toxicity of the compounds
Keksinnon mukaisten yhdisteiden akuuttia myrkylli-syyttå arvioitiin antamalla niita rotille laskimonsisai-sena ruiskeena. 3 urosrotan ryhmat saivat yhden ruiskeen 5 valiainetta tai laaketta (RS-15378 tai RS-26306) annok-sen ollessa 1,0 mg/kg. Kaksi muuta rottaryhmaa sai joko 0,3 mg tai 1,0 mg standardiyhdistetta (RS-68439)/kg.The acute toxicity of the compounds of the invention was assessed by intravenous injection to rats. Groups of 3 male rats received a single injection of 5 agents or drugs (RS-15378 or RS-26306) at a dose of 1.0 mg / kg. The other two groups of rats received either 0.3 mg or 1.0 mg of standard compound (RS-68439) / kg.
(Kaikki yhdisteet on nimetty aikaisemmissa esimerkeissa).(All compounds are named in the previous examples).
Ruiskeet annettiin hantålaskimoon 15-20 sekunnin 10 vålein. Elåimiå tarkkailtiin myrkyllisyyden kliinisten merkkien havaitsemiseksi valittdmasti antamisen jalkeen ja 0,5, 1,0, 3,0 ja 6,0 tunnin kuluttua ruiskeen antami-sesta.Injections were given into the dental vein every 10 to 20 seconds. Animals were monitored for clinical signs of toxicity at randomly after administration and at 0.5, 1.0, 3.0, and 6.0 hours after injection.
Tulokset on esitetty taulukossa 5.The results are shown in Table 5.
j » 56 9/326 3 (1) :3 Λ! -Η •Η »—( 3 (L) > Τ3 C Φ - (1)j »56 9/326 3 (1): 3 Λ! -Η • Η »- (3 (L)> Τ3 C Φ - (1)
•Η C S• Η C S
cn ή φ o ε -u oo 3 3 3 ^ (O -n >i :3 CO -P τιcn ή φ o ε -u oo 3 3 3 ^ (O -n> i: 3 CO -P τι
>i <D> i <D
-C X-C X
W >,W>,
3 -P P3 -P P
-PC Φ O :3 ε > :3 P in ro-PC Φ O: 3 ε>: 3 P in ro
3 -H -H3 -H -H
-P <H > (0 (0-P <H> (0 (0
tO -P -PtO -P -P
' ' M -P -P'' M -P -P
C tn 3 -Η Φ Φ •h >, > tn -p -p -p σ'-p a in in •H p -H C 3 3 (0 Λί 3 σ' -Η Η > > Ρ -Ρ 3 > ηΗ Φ Φ φ >ι Ο ·Η -Η S J .c κλ; w ωC tn 3 -Η Φ Φ • h>,> tn -p -p -p σ'-pa in in • H p -HC 3 3 (0 Λί 3 σ '-Η Η>> Ρ -Ρ 3> ηΗ Φ Φ φ> ι Ο · Η -Η SJ .c κλ; w ω
•Η r-I• Η r-I
ο — λ: σ' >ι Μ r-l \ σ> σ' -η ε c —' m ο ο ο φ - ^ ^ Φ ιη ο ι—ι *—ι ι—ι ι—i Ο >ι c a 5 0 a 3 φ c <#> _ ο Φ m ιη -Ρ * α . 0) >—I Ρ _Η —1 ·· : ·η 'Τ' Hr ^ ^ φ J Ό w ο νο X <Ν τ <Ν ·> 3 Ο * *τ5 *<ν“£ 5 - ^ο“ 5 * 5 Ja u_- 9 ζ Η α; ο 9 9 'oiS ο ί ^ 2 oamw-wo =3 3 Ρ σι Λ Λ 2* « 00 1 _L ^ Η Π3 «> I W CT UJ r-t > r-i 3 Μ ecoccrH Γ- artiraH OOHrth^a 3 to QO z T ι 1 m 4 ι <ac I ιο ' 9“ 9 9 =r ^ο - λ: σ '> ι Μ rl \ σ> σ' -η ε c - 'm ο ο ο φ - ^ ^ Φ ιη ο ι — ι * —ι ι — ι ι — i Ο> ι ca 5 0 a 3 φ c <#> _ ο Φ m ιη -Ρ * α. 0)> —I Ρ _Η —1 ··: · η 'Τ' Hr ^ ^ φ J Ό w ο νο X <Ν τ <Ν ·> 3 Ο * * τ5 * <ν “£ 5 - ^ ο“ 5 * 5 And u_- 9 ζ Η α; ο 9 9 'oiS ο ί ^ 2 oamw-wo = 3 3 Ρ σι Λ Λ 2 * «00 1 _L ^ Η Π3«> IW CT UJ rt> ri 3 Μ ecoccrH Γ- artiraH OOHrth ^ a 3 to QO z T ι 1 m 4 ι <ac I ιο '9 “9 9 = r ^
EnOiiD ^300 r-i SooaQ(N5.QQQ3Q * k · 57 9 :> 3 2 6EnOiiD ^ 300 r-i SooaQ (N5.QQQ3Q * k · 57 9:> 3 2 6
Fysiologiset formulatPhysiological formulas
Seuraavat ovat tyypillisiS fysiologisia koostumuk-sia, jotka sisaitavat vaikuttavana aineosana jotakin ta-mSn keksinnOn mukaista LHRH:n antagonistia, esimerkiksi 5 (N-Ac-D-Nal(2)1, D-pCl-Phe2, Pal(3)3'6, Bth8, D-Ala10)- LHRH:ta sellaisenaan tai fysiologisesti hyvåksyttavana suolanaan, esimerkiksi etikkahappoadditiosuolana, sinkki-suolana, sinkkitannaattisuola tms.The following are typical physiological compositions which contain as an active ingredient an LHRH antagonist according to the invention, for example 5 (N-Ac-D-Nal (2) 1, D-pCl-Phe2, Pal (3) 3 '). 6, Bth8, D-Ala10) - LHRH as such or as a physiologically acceptable salt thereof, for example, as an acetic acid addition salt, a zinc salt, a zinc tannate salt and the like.
A. Tablettiformulat 10 1. LHRH:n antagonistia 10,0 mgA. Tablet Formulations 1. LHRH antagonist 10.0 mg
Puristettavissa olevaa sokeria, USP 86,0 mgCompressible sugar, USP 86.0 mg
Kalsiumstearaattia 4,0 mg 2. LHRHrn antagonistia 10,0 mgCalcium stearate 4.0 mg 2. LHRH antagonist 10.0 mg
Puristettavissa olevaa sokeria, USP 88,5 mg 15 Magnesiumstearaattia 1,5 mg 3. LHRHrn antagonistia 5,0 mgCompressible sugar, USP 88.5 mg 15 Magnesium stearate 1.5 mg 3. LHRH antagonist 5.0 mg
Mannitolia, USP 83,5 mgMannitol, USP 83.5 mg
Magnesiumstearaattia, USP 1,5 mgMagnesium stearate, USP 1.5 mg
Esigelatinoitua tarkkelysta, USP 10,0 mg 20 4. LHRHrn antagonistia 10,0 mgPregelatinized observation, USP 10.0 mg 20 4. LHRH antagonist 10.0 mg
Laktoosia, USP 74,5 mgLactose, USP 74.5 mg
Esigelatinoitua tårkkelysta, USP 15,0 mgPregelatinized starch, USP 15.0 mg
Magnesiumstearaattia, USP 1,5 mg 25 Valmistusmenetelma (a) LHRHrn antagonist! liuotetaan riittflv&an maa-raan vetta, jotta muodostuu marka granulaatti, kun siihen sekoitetaan sokeriosuus tayteaineista. Taydellisen se-koittumisen jalkeen granulaatti kuivataan lautasella tai 30 leijukerroskuivurissa. Kuiva granulaatti seulotaan mah-dollisten suurten aggregaattien hajottamiseksi, ja sen jalkeen siihen sekoitetaan jaijelia olevat aineosat. Granulaatti puristetaan sitten tavanomaisella tabletointiko-neella maaratyn massan omaaviksi tableteiksi.Magnesium Stearate, USP 1.5 mg Method of Preparation (a) LHRH antagonist! dissolve in a sufficient amount of water to form a brandy granulate when mixed with a portion of the sugar excipients. After complete mixing, the granulate is dried on a plate or in a fluid bed dryer. The dry granulate is screened to break up any large aggregates and then mixed with the remaining ingredients. The granulate is then compressed into tablets having a defined mass on a conventional tableting machine.
35 (b) Tassa valmistusmenetelmasså kaikki formulat sisaltavat 0,01 % sisaitavat 0,01 % gelatiinia (USP). Ge- 58 92376 latiini liuotetaan ensin granulointiliuottimena toimivaan veteen ja sen jaikeen liuotetaan LHRH-analogi. Loput vai-heet on samanlalsla kuln edelia kohdassa (a).35 (b) In this method of preparation, all formulations contain 0.01% containing 0.01% gelatin (USP). Gel 58 92376 latin is first dissolved in water as a granulation solvent, and an LHRH analog is dissolved in its fraction. The remaining steps are at the same time as in (a).
Formulaa 4 voitaisiin kayttaa mytts suun kautta an- 5 nettavana tablettina.Formula 4 could be used as a mytts oral tablet.
B. Pitkåvaikutteinen lihaksensiséisesti injek- toitava formula 1. Pitkavaikutteinen llhaksensisMinen ruiske-sesamdljygeeli 10 LHR:n antagonistla 10,0 mgB. Long-acting Intramuscular Injection Formula 1. Long-acting Inhalation Sesame Diesel Gel 10 LHR Antagonist 10.0 mg
Alumiinimonostearaattia, USP 20,0 mgAluminum monostearate, USP 20.0 mg
SeesamiOljya gs ad 1,0 ml:ksiSeesamiOljya gs gives 1.0 ml
Alumiinimonostearaatti ja seesamiOljy yhdistetaan ja yhdistelmaa kuumennetaan 125°C:ssa samalla sekoittaen, 15 kunnes muodostuu kirkas keltainen liuos. Sen j&lkeen tSma seos autoklavoidaan steriloinnin suorittamiseksi ja sen annetaan jaahtya. Sitten lisataan LHRH-analogi aseptises-ti samalla trituroiden. Erityisen edullisia LHRH-analoge-ja ovat heikosti liukenevat suolat, esimerkiksi sinkki-20 suolat, sinkkitannaattisuolat, pamoaattisuolat ja vastaa-vat. Niilia on poikkeuksellisen pitka vaikutuksen kesto.The aluminum monostearate and sesame oil are combined and the combination is heated at 125 ° C with stirring until a clear yellow solution is formed. The tSma mixture is then autoclaved to perform sterilization and allowed to cool. The LHRH analog is then added aseptically while triturating. Particularly preferred LHRH analogs are sparingly soluble salts, for example, zinc-20 salts, zinc tannate salts, pamoate salts and the like. The Nile has an exceptionally long duration of action.
2. Pitkavaikutteinen lihaksensisainen ruiske -biologisesti hajoavasta polymeerista valmiste-tut mikrokapselit . 25 LHRH:n antagonistia 7 % 25/75 glykosidi/aktidikopolymeeria 93 % (ominaisviskositeetti 0,5)2. Long-acting intramuscular injection microcapsules made of biodegradable polymer. 25 LHRH antagonists 7% 25/75 glycoside / actide copolymer 93% (intrinsic viscosity 0.5)
Edelia olevan formulan mukaiset mikrokapselit sus-pendoidaan seuraavaan liuokseen: 30 Dekstroosia 5,0 % ; Natrium-CMC:ta 0,5 %The microcapsules of the above formula are suspended in the following solution: Dextrose 5.0%; 0.5% sodium CMC
Bentsyylialkoholia 0,9 %Benzyl alcohol 0.9%
Tween 80:a 0,1 %Tween 80 0.1%
Puhdistettua vetta gs 100,0 %:ksi 35 25 mg mikrokapseleita suspendoitiin 1,0 ml:aan vaiiainet- ta.Purified water to gs 100.0% 35 25 mg microcapsules were suspended in 1.0 ml of vehicle.
92326 59 C. Lihaksensisaisesti ruiskutettava vesiliuos LHRHrn antagonistia 0,5 %92326 59 C. Aqueous intramuscular injection of LHRH antagonist 0.5%
Etikkahappoa 0,02 mol/1Acetic acid 0.02 mol / l
Bentsyylialkanolia 0,9 % 5 Mannitolia 3,5 %Benzylalkanol 0.9% 5 Mannitol 3.5%
Propyleeniglykolia 20 %Propylene glycol 20%
NaOH:a pH:n saatamiseksi arvoon 5NaOH to bring the pH to 5
Vetta gs 100 %:ksiVetta gs to 100%
Etikkahappo, bentsyylialkoholl, mannitoll ja pro-10 pyleeniglykoli liuotettiin 90 %:iin vesimaarasta. Tahan liuokseen liuotettiin sitten LHRH:n antagonist!, ja pH saadettiin NaOHtlla. Lisattiin puuttuva vesimaara. Liuos suodatettiin 1 pm:n suodattimen lapi, pakattiin pieniin laakepulloihin ja steriloitiin autoklaavikasittelylia.Acetic acid, benzyl alcohol, mannitol and pro-10 methylene glycol were dissolved in 90% water. The LHRH antagonist was then dissolved in the paste solution, and the pH was adjusted with NaOH. The missing amount of water was added. The solution was filtered through a 1 μm filter, packaged in small vials and sterilized by autoclaving.
15 D. Nenaan annettava vesiformula LHRH:n antagonistia 50 mg 0,02M asetaattipuskuria 5 ml15 D. Nasal aqueous formula LHRH antagonist 50 mg 0.02M acetate buffer 5 ml
Natriumglykokolaattia 500 mg 0,02M asetaattipuskuria, pH 5,2 10 ml:ksi 20 E. Rektaalisesti annettava formulaSodium glycocholate 500 mg 0.02M acetate buffer, pH 5.2 to 10 ml 20 E. Formula for rectal administration
Perapuikko: LHRH:n antagonistia 5,0 mgSuppository: LHRH antagonist 5.0 mg
Witepsol H15:ta 20,0 g LHRH:n antagonisti yhdistettiin sulaan Witepsol .25 H15:een, ne sekoitettiin hyvin ja seos kaadettiin 2 g:n muotteihin.Witepsol H15 20.0 g of LHRH antagonist was combined with molten Witepsol .25 H15, mixed well and poured into 2 g molds.
··
Claims (10)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
FI930694A FI92211C (en) | 1987-02-05 | 1993-02-17 | Method for preparing therapeutically useful nonapeptide and decapeptide analogs that are antagonists of LHRH |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US1092387 | 1987-02-05 | ||
US07/010,923 US4801577A (en) | 1987-02-05 | 1987-02-05 | Nonapeptide and decapeptide analogs of LHRH useful as LHRH antagonists |
Publications (4)
Publication Number | Publication Date |
---|---|
FI880509A0 FI880509A0 (en) | 1988-02-04 |
FI880509A FI880509A (en) | 1988-08-06 |
FI92326B FI92326B (en) | 1994-07-15 |
FI92326C true FI92326C (en) | 1994-10-25 |
Family
ID=21748050
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
FI880509A FI92326C (en) | 1987-02-05 | 1988-02-04 | LHRH nonapeptide and decapeptide analogs useful as LHRH antagonists |
FI930694A FI92211C (en) | 1987-02-05 | 1993-02-17 | Method for preparing therapeutically useful nonapeptide and decapeptide analogs that are antagonists of LHRH |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
FI930694A FI92211C (en) | 1987-02-05 | 1993-02-17 | Method for preparing therapeutically useful nonapeptide and decapeptide analogs that are antagonists of LHRH |
Country Status (22)
Country | Link |
---|---|
US (2) | US4801577A (en) |
EP (1) | EP0277829B1 (en) |
JP (1) | JPH0791314B2 (en) |
KR (1) | KR930008095B1 (en) |
AT (1) | ATE109159T1 (en) |
AU (1) | AU614275B2 (en) |
BR (1) | BR1100688A (en) |
CA (1) | CA1339043C (en) |
DE (2) | DE3850789T2 (en) |
DK (1) | DK173544B1 (en) |
ES (1) | ES2056908T3 (en) |
FI (2) | FI92326C (en) |
HK (1) | HK35997A (en) |
HU (3) | HUT63855A (en) |
IE (1) | IE63707B1 (en) |
IL (1) | IL85316A (en) |
LU (1) | LU90657I2 (en) |
MX (1) | MX9202737A (en) |
NL (1) | NL300016I2 (en) |
NO (3) | NO176440C (en) |
NZ (1) | NZ223403A (en) |
ZA (1) | ZA88795B (en) |
Families Citing this family (45)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5688519A (en) * | 1987-04-06 | 1997-11-18 | Leonard; Robert J. | Flash-flow fused medicinal implants |
US4935491A (en) * | 1987-08-24 | 1990-06-19 | Board Of Regents, The University Of Texas System | Effective antagonists of the luteinizing hormone releasing hormone which release negligible histamine |
HU203204B (en) * | 1987-09-15 | 1991-06-28 | Sandoz Ag | Process for producing suppository containing calcitonin and taurocholic acid |
EP0312052B1 (en) * | 1987-10-15 | 1994-01-05 | Syntex (U.S.A.) Inc. | Intranasal administration of polypeptides in powdered form |
US5110904A (en) * | 1989-08-07 | 1992-05-05 | Abbott Laboratories | Lhrh analogs |
US5137669A (en) * | 1988-03-02 | 1992-08-11 | Endocon, Inc. | Manufacture of partially fused peptide pellet |
US5039660A (en) * | 1988-03-02 | 1991-08-13 | Endocon, Inc. | Partially fused peptide pellet |
WO1990010437A1 (en) * | 1989-03-09 | 1990-09-20 | Endocon, Inc. | Partially fused peptide pellet |
US6156731A (en) * | 1989-05-10 | 2000-12-05 | G. D. Searle & Co. | Polypeptide composition for oral administration |
US5232707A (en) * | 1989-07-10 | 1993-08-03 | Syntex (U.S.A.) Inc. | Solvent extraction process |
US5169932A (en) * | 1989-10-30 | 1992-12-08 | The Salk Institute For Biological Studies | Gnrh analogs |
US5084443A (en) * | 1989-12-04 | 1992-01-28 | Biomeasure, Inc. | Promoting expression of acetylcholine receptors with lhrh antagonist |
US5064939A (en) * | 1990-02-06 | 1991-11-12 | The Salk Institute For Biological Studies | Cyclic gnrh antagonists |
US5212288A (en) * | 1990-02-20 | 1993-05-18 | Syntex (U.S.A.) Inc. | Temporary minimal protection synthesis of serine-containing polypeptides |
EP0477499B1 (en) * | 1990-08-04 | 1994-01-26 | Hoechst Aktiengesellschaft | Gonadoliberin antagonists |
US5783562A (en) * | 1990-11-10 | 1998-07-21 | Asta Medica Aktiengesellschaft | Luteinizing hormone releasing hormone analogs |
CN1036343C (en) * | 1990-11-10 | 1997-11-05 | 天津市计划生育研究所 | Synthesis, products and its application for luleinizing hormone releasing hormone and antagonistic znalogue |
IL101074A (en) * | 1991-03-14 | 1997-09-30 | Salk Inst For Biological Studi | GnRH ANALOGS AND THEIR PREPARATION |
DE593491T1 (en) * | 1991-04-25 | 1994-11-17 | Romano S.-Cergue Deghenghi | LHRH antagonists. |
DE4117507A1 (en) * | 1991-05-24 | 1992-11-26 | Schering Ag | METHOD FOR PRODUCING N (ARROW HIGH) 6 (ARROW HIGH) SUBSTITUTED LYSINE DERIVATIVES |
WO1993003752A1 (en) * | 1991-08-19 | 1993-03-04 | The Arizona Board Of Regents On Behalf Of The University Of Arizona | Decapeptide having dopamine stimulating activity |
WO1993003751A1 (en) * | 1991-08-26 | 1993-03-04 | Abbott Laboratories | Compositions and methods for the sublingual or buccal administration of therapeutic agents |
US5487898A (en) * | 1991-08-26 | 1996-01-30 | Abbott Laboratories | Compositions and method for the sublingual or buccal administration therapeutic agents |
US5656727A (en) * | 1992-09-15 | 1997-08-12 | The Administrators Of The Tulane Educational Fund | Antagonists of LHRH |
US5480969A (en) * | 1992-09-15 | 1996-01-02 | The Administrators Of The Tulane Educational Fund | Antagonists of LHRH |
DE4305225A1 (en) * | 1993-02-19 | 1994-08-25 | Asta Medica Ag | New manufacturing process for Cetrorelix lyophilisate |
US6828415B2 (en) | 1993-02-19 | 2004-12-07 | Zentaris Gmbh | Oligopeptide lyophilisate, their preparation and use |
US5843901A (en) * | 1995-06-07 | 1998-12-01 | Advanced Research & Technology Institute | LHRH antagonist peptides |
US6043218A (en) | 1996-10-22 | 2000-03-28 | Medical University Of South Carolina | Positively charged non-natural amino acids, methods of making thereof, and use thereof in peptides |
US6566330B1 (en) * | 1996-10-22 | 2003-05-20 | Medical University Of South Carolina Foundation Research Development | Positively charged non-natural amino acids, methods of making and using thereof in peptides |
US5968895A (en) * | 1996-12-11 | 1999-10-19 | Praecis Pharmaceuticals, Inc. | Pharmaceutical formulations for sustained drug delivery |
DE19712718C2 (en) * | 1997-03-26 | 1999-09-23 | Asta Medica Ag | Immobilized and activity-stabilized complexes of LHRH antagonists and process for their preparation |
EP0994718B1 (en) | 1997-06-20 | 2005-08-17 | Akzo Nobel N.V. | Gonadotropin releasing hormone antagonist |
US6217844B1 (en) | 1998-04-27 | 2001-04-17 | Praecis Pharmaceuticals, Inc. | Methods for detecting lesions in dense breast tissue using LHRH antagonists |
US6455499B1 (en) | 1999-02-23 | 2002-09-24 | Indiana University Foundation | Methods for treating disorders associated with LHRH activity |
US6598784B2 (en) * | 2000-12-20 | 2003-07-29 | Meadwestvaco Packaging Syatens, Llc | Beverage carton with strap type carrying handle |
WO2002056903A2 (en) * | 2001-01-17 | 2002-07-25 | Praecis Pharmaceuticals Inc. | Methods for treating hormone associated conditions using a combination of lhrh antagonists and specific estrogen receptor modulators |
GB0320806D0 (en) | 2003-09-05 | 2003-10-08 | Astrazeneca Ab | Therapeutic treatment |
EP1711159B1 (en) | 2003-12-30 | 2013-03-20 | Durect Corporation | Solid implants containing a block copolymer for controlled release of a gnrh compound |
JP5361210B2 (en) * | 2008-02-22 | 2013-12-04 | キヤノン株式会社 | Resin composition and molded body |
CN102584945A (en) * | 2012-02-09 | 2012-07-18 | 深圳翰宇药业股份有限公司 | Preparation method for ganirelix acetate |
CN104231055A (en) * | 2013-06-18 | 2014-12-24 | 深圳翰宇药业股份有限公司 | Ganirelix precursor and method for preparing ganirelix acetate by using ganirelix precursor |
CN109443592A (en) * | 2018-09-19 | 2019-03-08 | 东华大学 | A kind of measuring method of temperature sensitive block copolymer lower critical solution temperature |
US20220267347A1 (en) | 2019-07-29 | 2022-08-25 | Sunshine Lake Pharma Co., Ltd. | Substituted pyrimidinedione compounds and uses thereof |
WO2023072284A1 (en) | 2021-11-01 | 2023-05-04 | 山东绿叶制药有限公司 | Gonadotropin-releasing hormone antagonist, and preparation method therefor and use thereof |
Family Cites Families (19)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4215038A (en) * | 1978-10-16 | 1980-07-29 | The Salk Institute For Biological Studies | Peptides which inhibit gonadal function |
US4419347A (en) * | 1980-10-06 | 1983-12-06 | Syntex (U.S.A.) Inc. | Nonapeptide and decapeptide analogs of LHRH, useful as LHRH antagonists |
US4341767A (en) * | 1980-10-06 | 1982-07-27 | Syntex Inc. | Nonapeptide and decapeptide analogs of LHRH, useful as LHRH antagonists |
US4667014A (en) * | 1983-03-07 | 1987-05-19 | Syntex (U.S.A.) Inc. | Nonapeptide and decapeptide analogs of LHRH, useful as LHRH antagonists |
US4581169A (en) * | 1982-12-21 | 1986-04-08 | Syntex (U.S.A.) Inc. | Nona-peptide and deca-peptide analogs of LHRH, useful as LHRH antagonists |
US4481190A (en) * | 1982-12-21 | 1984-11-06 | Syntex (U.S.A.) Inc. | Nonapeptide and decapeptide analogs of LHRH useful as LHRH antagonists |
FI832053L (en) * | 1982-06-10 | 1983-12-11 | Syntex Inc | NONAPEPTID- OCH DEKAPEPTIDANALOGER AV LHRH ANVAENDBARA SOM LHRH-ANTAGONISTER SAMT DERAS FRAMSTAELLNINGSFOERFARANDE |
ZA846154B (en) * | 1983-08-16 | 1985-03-27 | Akzo Nv | Lh-rh antagonists |
EP0145032B1 (en) * | 1983-08-16 | 1987-11-04 | Akzo N.V. | Lh- rh antagonists |
US4569927A (en) * | 1984-02-23 | 1986-02-11 | The Salk Institute For Biological Studies | GnRH Antagonists IV |
US4690916A (en) * | 1984-11-13 | 1987-09-01 | Syntex (U.S.A.) Inc. | Nona and decapeptide analogs of LHRH useful as LHRH antagonists |
EP0301850A3 (en) * | 1987-07-31 | 1990-10-31 | Syntex (U.S.A.) Inc. | Lhrh antagonist analogs and 19-nor-progestational steroids for therapy |
US5300492A (en) * | 1988-02-10 | 1994-04-05 | Tap Pharmaceuticals | LHRH analogs |
DE4117507A1 (en) * | 1991-05-24 | 1992-11-26 | Schering Ag | METHOD FOR PRODUCING N (ARROW HIGH) 6 (ARROW HIGH) SUBSTITUTED LYSINE DERIVATIVES |
US5480969A (en) * | 1992-09-15 | 1996-01-02 | The Administrators Of The Tulane Educational Fund | Antagonists of LHRH |
EP0683792B1 (en) * | 1992-12-18 | 2001-09-26 | Abbott Laboratories | Lhrh antagonists having modified aminoacyl residues at postions 5 and 6 |
US5502035A (en) * | 1993-08-06 | 1996-03-26 | Tap Holdings Inc. | N-terminus modified analogs of LHRH |
US5516759A (en) * | 1994-12-08 | 1996-05-14 | Tap Holdings Inc. | LHRH antagonists having lactam groups at the N-terminus |
US5574011A (en) * | 1995-04-04 | 1996-11-12 | Tien; Henry C. | Compositions and methods for the treatment of male-pattern baldness |
-
1987
- 1987-02-05 US US07/010,923 patent/US4801577A/en not_active Expired - Lifetime
-
1988
- 1988-02-04 HU HU921483A patent/HUT63855A/en unknown
- 1988-02-04 NO NO880494A patent/NO176440C/en not_active IP Right Cessation
- 1988-02-04 AT AT88300927T patent/ATE109159T1/en active
- 1988-02-04 IE IE29888A patent/IE63707B1/en not_active IP Right Cessation
- 1988-02-04 AU AU11265/88A patent/AU614275B2/en not_active Expired
- 1988-02-04 DK DK198800572A patent/DK173544B1/en not_active IP Right Cessation
- 1988-02-04 ES ES88300927T patent/ES2056908T3/en not_active Expired - Lifetime
- 1988-02-04 DE DE3850789T patent/DE3850789T2/en not_active Expired - Lifetime
- 1988-02-04 EP EP88300927A patent/EP0277829B1/en not_active Expired - Lifetime
- 1988-02-04 JP JP63026418A patent/JPH0791314B2/en not_active Expired - Lifetime
- 1988-02-04 DE DE2000175027 patent/DE10075027I2/en active Active
- 1988-02-04 ZA ZA88795A patent/ZA88795B/en unknown
- 1988-02-04 HU HU19/88A patent/HU215855B/en unknown
- 1988-02-04 IL IL85316A patent/IL85316A/en not_active IP Right Cessation
- 1988-02-04 CA CA000558099A patent/CA1339043C/en not_active Expired - Lifetime
- 1988-02-04 NZ NZ223403A patent/NZ223403A/en unknown
- 1988-02-04 FI FI880509A patent/FI92326C/en active IP Right Grant
- 1988-02-05 KR KR1019880001047A patent/KR930008095B1/en not_active IP Right Cessation
-
1992
- 1992-06-08 MX MX9202737A patent/MX9202737A/en unknown
- 1992-07-03 NO NO922632A patent/NO301014B1/en not_active IP Right Cessation
-
1993
- 1993-02-17 FI FI930694A patent/FI92211C/en active Protection Beyond IP Right Term
- 1993-06-02 US US08/526,940 patent/US5767082A/en not_active Expired - Lifetime
-
1995
- 1995-02-17 HU HU95P/P00080P patent/HU210819A9/en unknown
-
1997
- 1997-03-20 HK HK35997A patent/HK35997A/en not_active IP Right Cessation
- 1997-05-08 BR BR1100688-9A patent/BR1100688A/en active IP Right Grant
-
2000
- 2000-09-04 NL NL300016C patent/NL300016I2/en unknown
- 2000-10-18 LU LU90657C patent/LU90657I2/en unknown
-
2001
- 2001-01-16 NO NO2001001C patent/NO2001001I1/en unknown
Also Published As
Similar Documents
Publication | Publication Date | Title |
---|---|---|
FI92326C (en) | LHRH nonapeptide and decapeptide analogs useful as LHRH antagonists | |
US4667014A (en) | Nonapeptide and decapeptide analogs of LHRH, useful as LHRH antagonists | |
JP3645255B1 (en) | GnRH antagonists modified at positions 5 and 6 | |
EP0097031B1 (en) | Nonapeptide and decapeptide analogs of lhrh useful as lhrh antagonists, their preparation and compositions containing them | |
AU593764B2 (en) | Nonapeptide and decapeptice analogs of LHRH, useful as LHRH agonists | |
CA2022444C (en) | Lhrh analogs | |
US4481190A (en) | Nonapeptide and decapeptide analogs of LHRH useful as LHRH antagonists | |
JP4191259B2 (en) | GnRH antagonist | |
AU594652B2 (en) | Nona and decapeptide analogs of LHRH useful as LHRH antagonists | |
WO1995004540A1 (en) | N-terminus modified analogs of lhrh | |
KR0123009B1 (en) | Gnrh analogs | |
EP0328090A2 (en) | LHRH analogs | |
US4581169A (en) | Nona-peptide and deca-peptide analogs of LHRH, useful as LHRH antagonists | |
US5698522A (en) | 6-position modified decapeptide LHRH antagonists | |
EP0683792A1 (en) | Lhrh antagonists having modified aminoacyl residues at postions 5 and 6 | |
US5508383A (en) | Cyclic peptide LHRH antagonists | |
EP0301850A2 (en) | LHRH antagonist analogs and 19-nor-progestational steroids for therapy | |
HU195234B (en) | Process for production of nonapeptide and dekapeptide hormone-analogs freeing lutheining hormones with antagonizing hormone effect and medical preparatives containing them |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
BB | Publication of examined application | ||
FG | Patent granted |
Owner name: SYNTEX (U.S.A.) INC. |